Tumor-associated Vasculature Research Articles

A Wurster-Type Covalent Organic Framework with Internal Electron Transfer-Enhanced Catalytic Capacity for Tumor Therapy.

The low immunogenicity of tumors, along with the abnormal structural and biochemical barriers of tumor-associated vasculature, impedes the infiltration and function of effector T cells at the tumor site, severely inhibiting the efficacy of antitumor immunotherapy. In this study, a cobaloxime catalyst and STING agonist (MSA-2)-coloaded Wurster-type covalent organic framework (Co-TB COF-M) with internal electron transfer-enhanced catalytic capacity was developed as a COF-based immune activator. The covalently anchored cobaloxime adjusts the energy band structure of TB COF and provides it with good substrate adsorption sites, enabling it to act as an electron transmission bridge between the COF and substrate in proton reduction catalytic reactions. This property significantly enhances the sonodynamic catalytic performance. Under sono-irradiation, Co-TB COF-M can produce a substantial amount of reactive oxygen species (ROS) to induce Gasdermin D-mediated pro-inflammatory pyroptosis, thereby effectively enhancing the immunogenicity of tumors. Furthermore, MSA-2 is specifically released in response to ROS at the tumor site, minimizing the off-target side effects. More importantly, Co-TB COF-induced STING activation normalizes tumor vasculature and increases the expression of endothelial T cell adhesion molecules, which greatly enhance the infiltration and function of effector T cells. Thus, Co-TB COF-M as an immune activator could remold the tumor microenvironment, leading to increased infiltration and an improved function of T cells for immunotherapy.

Far-Red Aggregation-Induced Emission Hydrogel-Reinforced Tissue Clearing for 3D Vasculature Imaging of Whole Lung and Whole Tumor.

Understanding the vascular formation and distribution in metastatic lung tumors is a significant challenge due to autofluorescence, antibody/dye diffusion in dense tumor, and fluorophore stability when exposed to solvent-based clearing agents. Here, an approach is presented that redefines 3D vasculature imaging within metastatic tumor, peritumoral lung tissue, and normal lung. Specifically, a far-red aggregation-induced emission nanoparticle with surface amino groups (termed as TSCN nanoparticle, TSCNNP) is designed for in situ formation of hydrogel (TSCNNP@Gel) inside vasculatures to provide structural support and enhance the fluorescence in solvent-based tissue clearing method. Using this TSCNNP@Gel-reinforced tissue clearing imaging approach, the critical challenges are successfully overcome and comprehensive visualization of the whole pulmonary vasculature up to 2µm resolution is enabled, including its detailed examination in metastatic tumors. Importantly, features of tumor-associated vasculature in 3D panoramic views are unveiled, providing the potential to determine tumor stages, predict tumor progression, and facilitate the histopathological diagnosis of various tumor types.

AI-based radiomics model for predicting immune checkpoint inhibitor–related pneumonitis (CIP) in patients with advanced NSCLC: An external validation study.

12136 Background: The growing implementation of immunotherapy in advanced non-small cell lung cancer (NSCLC) management has led to an increase in adverse events, notably immune checkpoint inhibitor-related pneumonitis (CIP). CIP, a significant and potentially fatal complication, often necessitates the discontinuation of immunotherapy, thereby impacting patient outcomes severely. The absence of biomarkers for early detection and management of CIP represents an urgent and unmet clinical need. This expanded study explores the potential of Artificial Intelligence (AI) algorithms in predicting CIP in NSCLC patients undergoing immunotherapy from pre-treatment CT scans. Methods: A cohort of 220 stage III-IV NSCLC patients receiving immunotherapy was considered. The patients were divided into a training set (D1, n=105, Institution A), an internal validation set (D2, n=45, Institution A), and an external validation set (D3, N=70, Institutions B and C). Manual delineation of the tumor on baseline CT was performed by three physicians working in consensus. The Picture Health Px platform was employed for AI-powered deep phenotyping of the tumor and its surrounding habitat, including segmentation of the tumor associated vasculature and featurization. Quantitative features relating to the twistedness of the tumor-associated vasculature and tumoral heterogeneity patterns were extracted. These features were used to train a neural network classifier for CIP prediction. Weighting techniques during training were used to compensate for the rarity of CIP. A threshold for CIP classification was set within D1 and applied to the testing sets. Results: 43.6% of patients received an immunotherapy-only regimen and 56.4% received combined immunochemotherapy. Of these, 18.2.% experienced pneumonitis events, with 50.0% being CIP. The CIP subgroup had 50.0% grade 1 CIP, 30.0% grade 2, and 20.0% grade 3. The cross-validated AUC on D1 was 0.76 (95% CI: 0.62-0.90). The AUC was 0.61, 0.62, and 0.64, respectively, on D2, D3, and all validation data combined (D2+D3). The model correctly identified 71.4% of grade 1/2 CIP events and 33.3% of grade 3 CIP, with a corresponding false positive rate of 33.0%. Severe/grade 3 IO-related pneumonitis prediction was likely limited by the small number of severe cases from the training institution (n=1). Conclusions: We demonstrated a radiomic AI signature could identify patients at risk of CIP prior to treatment across multiple external institutions. This approach could improve the identification and management of CIP in NSCLC patients on immunotherapy, thereby enhancing patient outcomes. Expanded training and validation to account for rare, but possibly fatal, high grade CIPs is needed to expand the clinical benefit of radiologic predictors of pneumonitis to the most severe cases.

An interpretable AI-derived radiology signature to identify patients at risk of progression on the PACIFIC regimen for unresectable non-small cell lung cancer.

8079 Background: Advances in the treatment of unresectable NSCLC have emerged through the combination of chemotherapy, radiotherapy, and immune checkpoint inhibitors (ICI), also known as the PACIFIC regimen. Despite this protocol’s benefits, it lacks predictive biomarkers and many patients will ultimately fail to respond. Artificial intelligence (AI) has shown promise in identifying patients responsive to ICI, but its ability to identify patients who may not benefit from such combination regimens remains underexplored. We introduce an AI-enabled approach leveraging interpretable imaging biomarkers, such as tumor heterogeneity and twistedness of the tumor-associated vasculature, to identify patients who will fail to benefit from chemo-radio-immunotherapy (CRIT) using pretreatment scans. Methods: We analyzed CT data from 148 NSCLC patients with predominantly stage III (91%) disease from two institutions. Target lesions were delineated by a trained radiologist. The Picture Health Px platform was utilized to segment the lungs and pulmonary vessels. Subsequently, a number of interpretable imaging features from the tumor and surrounding tissues were extracted. A Cox proportional hazards model of feature clusters was developed on the training set (n = 101) to stratify patients into benefit groups associated with progression-free survival (PFS). A cohort of 47 patients receiving CRIT were held out for testing. Results: The imaging-derived high-risk group defined by the model was associated with decreased PFS in the test set, with a hazard ratio (HR) of 4.99 (95% CI: 2.04-12.18; p < 0.005) and concordance index (C-index) of 0.66. The risk groups identified by the model outperformed PD-L1 negative status in identifying likely progressors (C-index = 0.52; HR = 1.49 [0.51-4.54], p = 0.47), and were additionally independently prognostic of PD-L1 status when compared in a multivariable analysis (p < 0.001). The primary features driving the model were measurements of the radius and tortuosity of the tumor-associated vasculature, as well as heterogeneity metrics of the tumor. Conclusions: An AI imaging tool that stratifies patients by risk after CRIT from baseline radiology was developed. The tool was able to strongly identify a subset of patients at very high risk of progression if treated with CRIT. These risk groups outperformed PD-L1 and thus may address biomarker gaps in the CRIT setting. With further clinical validation, radiology-based risk stratification could be used to identify a subset of unresectable NSCLC patients for whom alternatives to the standard of care should be explored.

Abstract 7006: Identification of endothelial-cell-specific DNA methylation alterations in breast cancer with deconvolution of the tumor microenvironment

Abstract The tumor microenvironment (TME) plays a pivotal role in cancer progression, with tumor endothelial cells (TECs) emerging as key orchestrators of angiogenesis and vascular remodeling. Tumor-associated vasculature can have increased permeability and irregular blood flow, impacting drug delivery and tumor immune response. Understanding the molecular alterations within TECs may help identify targeted therapies and improve treatment outcomes with existing therapies. We combined TME cell-type deconvolution with modeling to identify cell-specific methylation alterations of TECs in breast cancer. Genome-scale DNA methylation array data accessed in GEO and GTEx from preinvasive, mixed, and invasive breast cancer tissue (n=609) & normal breast tissue (n=230). We applied HiTIMED, a reference-based cell-type deconvolution algorithm, to estimate endothelial and other cell-type proportions in the TME. Next, we applied CellDMC, a statistical algorithm that identifies cell-specific driven differential methylation, to our cell fraction results, unraveling differentially methylated positions occurring in the endothelial compartment of the angiogenic component of our breast tissue DNA methylation profiles. We identified 29 TEC driven differentially methylated positions (FDR ≤ 8e-03), including loci within genes in angiogenic pathways regulated by VEGF, endothelial inflammation and activation genes, and tumor-associated vasculature factors in the TME. Our findings suggest that variation in DNA methylation between TECs and normal endothelial cells is crucial to understanding the TME and providing insights into unknown cancer cell behaviors that contribute to tumor growth and metastasis, thus leading to the design of novel efficient therapies that target the malignant vascular component. Our study demonstrates the power of combining cell-type deconvolution and a modeling framework that identifies cell-specific somatic alterations to unravel the intricate characteristics of TECs in breast cancer. Furthermore, this study highlights the potential of DNA methylation profiling as a robust tool for characterizing TEC heterogeneity and elucidating the molecular basis of angiogenesis beyond breast tissue in the context of diverse cancer types. Our integrative approach holds promise for advancing precision oncology, paving the way for effective and targeted therapies aimed at modulating the tumor vasculature to impede cancer progression in breast cancer and other cancer types. Citation Format: Barbara Karakyriakou, Ze Zhang, Irma Vlasac, Pauline Mochama, Lucas Salas, Brock Christensen. Identification of endothelial-cell-specific DNA methylation alterations in breast cancer with deconvolution of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7006.

Abstract 912: CheckpointPx, an interpretable radiology AI tool, predicts checkpoint blockade benefit independent of PDL1 status in non-small cell lung cancers (NSCLC): A multi-institutional validation study

Abstract Background: Immune Checkpoint Inhibitors (ICIs) are a cornerstone in the treatment of cancers such as Non-Small Cell Lung Cancer (NSCLC). Despite the standard use of biomarkers such as PD-L1 expression for selecting ICI regimens, recent studies suggest missed opportunities for benefit in PD-L1 low/negative patients. Therefore, there is an urgent need for new biomarker strategies capable of guiding the use of ICIs in NSCLC. We introduce and validate an AI-powered radiology tool for ICI patient selection that provides interpretable predictions by quantifying aspects of tumor biology on baseline CT scans directly associated with ICI efficacy, such as tumor heterogeneity and twistedness of the tumor-associated vasculature. Methods: We analyzed CT scans of 439 patients treated with ICI at 4 institutions (D1-D4) using the Picture Health Px platform. Experienced radiologists and physicians across institutions delineated target lesions on baseline CT scans. A deep learning model was used to segment pulmonary vessels. CheckpointPx v1.11, an interpretable neural network incorporating quantitative features extracted from within the tumor and its vasculature, was trained to predict ICI response (defined as disease control per RECIST best overall response) on n=247 patients (D1-D3). It was assessed with respect to response and progression free-survival (PFS) on 192 patients (D2-D4), with D4 (n=105) being an external validation set. CheckpointPx was also evaluated within PDL1 subsets among testing set patients where available (n=138). Results: The cohort was predominantly late stage (Stage 3/4) (>85%) and was mixed with respect to ICI line of treatment (1st-4th). 122 of 247 (49%) and 119 of 192 (62%) were responders in training and test cohorts respectively. CheckpointPx included 16 features, such as entropy-based heterogeneity and quantitative vessel tortuosity (twistedness). The model predicted response to ICI with an AUC=0.65 on the test set. The CheckpointPx High Risk group significantly stratified patients by PFS (HR=1.67 [1.22-2.28], p=0.001). This separation remained significant within the subset of PDL1-negative (HR=2.71 [1.35-5.44], p=0.005) and PDL1-positive patients (HR=2.05 [1.22-3.46], p=0.007). Conclusions: From baseline radiology, CheckpointPx was shown to strongly predict ICI outcomes across multiple institutions, NSCLC stages, and lines of ICI. Furthermore, the tool stratified patients by ICI benefit within both PDL1-positive and negative subsets - suggesting the potential of CheckpointPx to address critical gaps in the NSCLC biomarker landscape. CheckpointPx is driven by interpretable imaging biomarkers, and thus its predictions are tied to specific and quantifiable phenotypic attributes of the tumor and its microenvironment. Citation Format: Amogh Hiremath, Seyoung Lee, Jeeyeon Lee, Peter Haseok Kim, Kai Zhang, Salie Lee, Monica Yadav, Maria Jose Aguilera Chuchuca, Taegyu Um, Myungwoo Nam, Liam Il-Young Chung, Hye Sung Kim, Jisang Yu, Trie Arni Djunadi, Leeseul Kim, Youjin Oh, Sungmi Yoon, Zunairah Shah, Yuchan Kim, Ilene Hong, Grace Kang, Jessica Jang, Amy Cho, Soowon Lee, Cecilia Nam, Timothy Hong, Yury S. Velichko, Amit Gupta, Vamsidhar Velcheti, Anant Madabhushi, Nathaniel Braman, Young Kwang Chae. CheckpointPx, an interpretable radiology AI tool, predicts checkpoint blockade benefit independent of PDL1 status in non-small cell lung cancers (NSCLC): A multi-institutional validation study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 912.

Abstract 3941: Wild-type KRAS dosage in mutant KRAS lung cancer: Implications for tumorigenesis and therapeutic response

Abstract Lung adenocarcinomas (LUAD) are frequently driven by activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS). Previous studies have suggested that wild-type (wt)-RAS signaling may modulate cancer progression and drug resistance in these tumors. However, the interplay between oncogenic KRAS and wt-RAS-like GTPases in lung cancer biology and treatment remains debated. Here, we investigated the impact of increased wt-KRAS dosage in LUAD harboring mutant KRAS on lung cancer phenotypes and response to anti-KRAS therapies. Increased wt-KRAS dosage occurs in LUAD due to the loss of LZTR1, a proteostatic regulator of RAS-like GTPases. LZTR1 mutations, deletions, or downregulation co-occur with oncogenic KRAS mutations in more than 40% of LUAD patients. Using a global protein stability screen, we identified LZTR1 as the main regulator of the stability of wt-KRAS, but not of the oncogenic KRAS mutants. Proteomic analysis of LUAD cells confirmed that LZTR1 loss leads to elevated expression of wt-KRAS, but not of KRAS-G12D, indicating that LZTR1 loss affects wt-RAS rather than mutant KRAS signaling. LZTR1 loss in LUAD patients is associated with poor prognosis and hypoxic expression signatures. In a mouse model of Kras-mutated LUAD, Lztr1 haploinsufficiency promoted tumor initiation and progression. Moreover, Lztr1 loss results in hypoxic tumors with inflamed, tortuous, insufficiently perfused, and leaky vessels. Integrative proteomic and phosphoproteomic analysis revealed increased RAL/TBK1 pathway activity in LZTR1-depleted lung cancer cells and Lztr1-deleted tumors. This leads to enhanced secretion of proangiogenic and proinflammatory factors, such as VEGF and TNFα, by LZTR1-deficient cells and an increased number of neutrophils in Lztr1-deleted tumors. TNF-activated neutrophils present at vascular branching points can modulate vascular remodeling by influencing the formation and function of blood vessels. Pharmacological inhibition of RAL/TBK1 signaling with amlexanox, an anti-NF-κB/TBK1 drug, reduces TNFα secretion, decreases the number of neutrophils, and normalizes the tumor-associated vasculature. We demonstrated that increased wt-KRAS dosage, but not other small GTPases, mimicked the effects of LZTR1 loss on RAL/TBK1 signaling and VEGF secretion, suggesting that LZTR1 loss alters the heterogeneity of KRAS-mutated lung cancer by modulating wt-KRAS levels. Furthermore, MRTX1133 a selective inhibitor of KRAS-G12D, showed limited effect in the Kras-G12D, Lztr1-loss LUAD model, whereas the combination of MRTX1133 and amlexanox suppress tumor growth, likely due to the restoration of the tumor vasculature by RAL/TBK1 inhibition. Our study reveals novel insights into the role of wt-KRAS dosage in mutant KRAS lung cancer and identifies potential therapeutic strategies to target patients with dysregulated KRAS proteostasis due to LZTR1 loss. Citation Format: Tonci Ivanisevic, Greetje Vande Velde, Peihua Zhao, Wout Magits, Raj N. Sewduth, Anna A. Sablina. Wild-type KRAS dosage in mutant KRAS lung cancer: Implications for tumorigenesis and therapeutic response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3941.

Low- and High-Grade Glioma-Associated Vascular Cells Differentially Regulate Tumor Growth.

A key feature distinguishing high-grade glioma (HG) from low-grade glioma (LG) is the extensive neovascularization and endothelial hyperproliferation. Prior work has shown that tumor-associated vasculature from HG is molecularly and functionally distinct from normal brain vasculature and expresses higher levels of protumorigenic factors that promote glioma growth and progression. However, it remains unclear whether vessels from LG also express protumorigenic factors, and to what extent they functionally contribute to glioma growth. Here, we profile the transcriptomes of glioma-associated vascular cells (GVC) from IDH-mutant (mIDH) LG and IDH-wild-type (wIDH) HG and show that they exhibit significant molecular and functional differences. LG-GVC show enrichment of extracellular matrix-related gene sets and sensitivity to antiangiogenic drugs, whereas HG-GVC display an increase in immune response-related gene sets and antiangiogenic resistance. Strikingly, conditioned media from LG-GVC inhibits the growth of wIDH glioblastoma cells, whereas HG-GVC promotes growth. In vivo cotransplantation of LG-GVC with tumor cells reduces growth, whereas HG-GVC enhances tumor growth in orthotopic xenografts. We identify ASPORIN (ASPN), a small leucine-rich repeat proteoglycan, highly enriched in LG-GVC as a growth suppressor of wIDH glioblastoma cells in vitro and in vivo. Together, these findings indicate that GVC from LG and HG are molecularly and functionally distinct and differentially regulate tumor growth. Implications: This study demonstrated that vascular cells from IDH-mutant LG and IDH-wild-type HG exhibit distinct molecular signatures and have differential effects on tumor growth via regulation of ASPN-TGFβ1-GPM6A signaling.

Expression of PSMA in Tumor-Associated Vasculature Predicts Poorer Survival in Patients With Hepatocellular Carcinoma and Is Likely Associated With PD-L1.

PSMA (prostate-specific membrane antigen) is a type II transmembrane glycoprotein recently found to be expressed in hepatocellular carcinoma (HCC). We aimed to characterize the expression pattern of PSMA in HCC and its association with clinicopathologic parameters and other biomarkers. Immunohistochemical studies for PSMA were performed on a previously established tissue microarray of 103 surgically resected HCC. Conceivable PSMA expression in ≥5% tumor-associated vasculature (TAV) was considered positive, and was identified in 56 (54.4%) tumors. Eight (7.8%) tumors also showed membranous/cytoplasmic and/or canalicular staining in tumor cells. By chi-square tests, only PSMA-positive TAV was associated with moderate-to-poorly differentiated HCC and the modified higher tumor stage (P < .05). PSMA-positive TAV was not associated with age, sex, or expression of glypican-3, keratin 7, CD3, CD8, HHLA-2, but marginally correlated with programmed death-ligand 1 (PD-L1) expression (P = .052). Kaplan-Meier survival analysis revealed PSMA-positive TAV as an independent risk factor for poorer disease-specific survival (P = .008). Co-expression of PD-L1 did not ameliorate the adverse prognostication of PSMA-positive TAV. Membranous/cytoplasmic/canalicular expression of PSMA alone was not prognostically significant. Our study confirmed that PSMA-positive TAV is a prospective diagnostic and prognostic biomarker for HCC. Co-expression of PSMA with PD-L1 may suggest potential crosstalk between the 2 proteins, likely regulating the tumor microenvironment.

PEDF-Enriched Extracellular Vesicle for Vessel Normalization to Potentiate Immune Checkpoint Blockade Therapy

The abnormal tumor vasculature acts as the physical and functional barrier to the infiltration and activity of effector T cells, leading to the low response rate of immune checkpoint inhibitors (ICIs). Herein, antiangiogenic extracellular vesicles that enable normalization of the tumor-associated vasculature were prepared to potentiate the efficacy of ICIs. Small extracellular vesicles were exploited as the delivery platform to protect the antiangiogenic protein, pigment epithelium-derived factor (PEDF), from proteolytic degradation. Along with the physicochemical characteristics of the PEDF-enriched extracellular vesicles (P-EVs), their inhibitory effects on migration, proliferation, and tube formation of endothelial cells were investigated in vitro. In tumor-bearing mice, it was confirmed that, compared to bare PEDFs, P-EVs efficiently reduced vessel leakiness, improved blood perfusion, and attenuated hypoxia. Consequently, when combined with anti-PD-1 antibodies, P-EVs remarkably augmented the antitumor immunity, as evidenced by increased infiltration of CD8 + T cells and reduced regulatory T cells. These results suggest that P-EVs are promising therapeutics for tumors refractory to ICIs.

The in situ transcriptomic landscape of breast tumour-associated and normal adjacent endothelial cells

Background and aimsTriple Negative Breast Cancer (TNBC) is associated with increased angiogenesis, which is known to aid tumour growth and metastasis. Anti-angiogenic therapies that have been developed to target this feature have mostly generated disappointing clinical results. Further research into targeted approaches is limited by a lack of understanding of the in situ molecular profile of tumour-associated vasculature. In this study, we aimed to understand the differences in the molecular profiles of tumour endothelial cells vs normal-adjacent endothelial cells in TNBC tissues. MethodWe have applied unbiased whole transcriptome spatial profiling of in situ gene expressions of endothelial cells localized in full-face patient TNBC tissues (n = 4) and normal-adjacent regions of the same patient breast tissues. ResultsOur comparative analysis revealed that 2412 genes were differentially expressed (padj < 0.05) between the tumour endothelial cells and normal-adjacent endothelial cells. Pathway enrichment showed the enrichment of gene sets related to cell-cell, cell-ECM adhesion, chromatin organization and remodeling, and protein-DNA complex subunit organization. ConclusionOverall, the results revealed unique molecular profiles and signalling pathways of tumour-associated vasculature, which is a critical step towards larger cohort studies investigating potential targets for TNBC prognosis and anti-angiogenic treatments.

B7H3 targeting gold nanocage pH-sensitive conjugates for precise and synergistic chemo-photothermal therapy against NSCLC

BackgroundThe combination of drug delivery with immune checkpoint targeting has been extensively studied in cancer therapy. However, the clinical benefit for patients from this strategy is still limited. B7 homolog 3 protein (B7-H3), also known as CD276 (B7-H3/CD276), is a promising therapeutic target for anti-cancer treatment. It is widely overexpressed on the surface of malignant cells and tumor vasculature, and its overexpression is associated with poor prognosis. Herein, we report B7H3 targeting doxorubicin (Dox)-conjugated gold nanocages (B7H3/Dox@GNCs) with pH-responsive drug release as a selective, precise, and synergistic chemotherapy-photothermal therapy agent against non-small-cell lung cancer (NSCLC).ResultsIn vitro, B7H3/Dox@GNCs exhibited a responsive release of Dox in the tumor acidic microenvironment. We also demonstrated enhanced intracellular uptake, induced cell cycle arrest, and increased apoptosis in B7H3 overexpressing NSCLC cells. In xenograft tumor models, B7H3/Dox@GNCs exhibited tumor tissue targeting and sustained drug release in response to the acidic environment. Wherein they synchronously destroyed B7H3 positive tumor cells, tumor-associated vasculature, and stromal fibroblasts.ConclusionThis study presents a dual-compartment targeted B7H3 multifunctional gold conjugate system that can precisely control Dox exposure in a spatio-temporal manner without evident toxicity and suggests a general strategy for synergistic therapy against NSCLC.

3D microengineered vascularized tumor spheroids for drug delivery and efficacy testing.

Tumor angiogenesis is regarded as a promising target for limiting cancer progression because tumor-associated vasculature supplies blood and provides a path for metastasis. Thus, in vitro recapitulation of vascularized tumors is critical to understand the pathology of cancer and identify the mechanisms by which tumor cells proliferate, metastasize, and respond to drugs. In this study, we microengineered a vascularized tumor spheroid (VTS) model to reproduce the pathological features of solid tumors. We first generated tumor-EC hybrid spheroids with self-assembled intratumoral vessels, which enhanced the uniformity of the spheroids and peritumoral angiogenic capacity compared to spheroids composed only with cancer cells. Notably, the hybrid spheroids also exhibited expression profiles associated with aggressive behavior. The blood vessels sprouting around the hybrid spheroids on the VTS chip displayed the distinctive characteristics of leaky tumor vessels. With the VTS chip showing a progressive tumor phenotype, we validated the suppressive effects of axitinib on tumor growth and angiogenesis, which depended on exposure dose and time, highlighting the significance of tumor vascularization to predict the efficacy of anticancer drugs. Ultimately, we effectively induced both lymphangiogenesis and angiogenesis around the tumor spheroid by promoting interstitial flow. Thus, our VTS model is a valuable platform with which to investigate the interactions between tumor microenvironments and explore therapeutic strategies in cancer. STATEMENT OF SIGNIFICANCE: We conducted an integrative study within a vascularized tumor spheroid (VTS) model. We first generated tumor-EC hybrid spheroids with self-assembled intratumoral vessels, which enhanced the uniformity of the spheroids and peritumoral angiogenic capacity compared to spheroids composed only with cancer cells. Through RNA sequencing, we elucidated that the tumor-EC hybrid spheroids exhibited expression profiles associated with aggressive behavior such as cancer progression, invasion and metastasis. The blood vessels sprouting around the hybrid spheroids on the VTS chip displayed the distinctive characteristics of leaky tumor vessels. We further validated the suppressive effects of axitinib on tumor growth and angiogenesis, depending on exposure dose and time. Ultimately, we effectively induced both lymphangiogenesis and angiogenesis around the tumor spheroid by promoting interstitial flow.

Molecular Mechanisms of Chloroquine and Hydroxychloroquine Used in Cancer Therapy

Tumour relapse, chemotherapy resistance, and metastasis continue to be unsolved issues in cancer therapy. A recent approach has been to scrutinise drugs used in the clinic for other illnesses and modify their structure to increase selectivity to cancer cells. Chloroquine (CQ) and hydroxychloroquine (HCQ), known antimalarials, have successfully treated autoimmune and neoplastic diseases. CQ and HCQ, well-known lysosomotropic agents, induce apoptosis, downregulate autophagy, and modify the tumour microenvironment. Moreover, they affect the Toll 9/NF-κB receptor pathway, activate stress response pathways, enhance p53 activity and CXCR4-CXCL12 expression in cancer cells, which would help explain their effects in cancer treatment. These compounds can normalise the tumour associated vasculature, promote the activation of the immune system, change the phenotype of tumour-associated macrophages (from M2 to M1), and stimulate cancer-associated fibroblasts. We aim to review the historical aspects of CQ and its derivatives and the most relevant mechanisms that support the therapeutic use of CQ and HCQ for the treatment of cancer.

STING-activating nanoparticles normalize the vascular-immune interface to potentiate cancer immunotherapy.

The tumor-associated vasculature imposes major structural and biochemical barriers to the infiltration of effector T cells and effective tumor control. Correlations between stimulator of interferon genes (STING) pathway activation and spontaneous T cell infiltration in human cancers led us to evaluate the effect of STING-activating nanoparticles (STANs), which are a polymersome-based platform for the delivery of a cyclic dinucleotide STING agonist, on the tumor vasculature and attendant effects on T cell infiltration and antitumor function. In multiple mouse tumor models, intravenous administration of STANs promoted vascular normalization, evidenced by improved vascular integrity, reduced tumor hypoxia, and increased endothelial cell expression of T cell adhesion molecules. STAN-mediated vascular reprogramming enhanced the infiltration, proliferation, and function of antitumor T cells and potentiated the response to immune checkpoint inhibitors and adoptive T cell therapy. We present STANs as a multimodal platform that activates and normalizes the tumor microenvironment to enhance T cell infiltration and function and augments responses to immunotherapy.

Human intravital microscopy in the study of sarcomas: an early trial of feasibility.

ClinicalTrials.gov, identifier NCT03517852; ClinicalTrials.gov, identifier NCT03823144.

Abstract 5569: Quantification of tumor-associated vasculature as an imaging biomarker for monitoring the response of triple-negative breast cancer to neoadjuvant chemotherapy

Abstract Introduction: Patients with locally advanced, triple-negative breast cancer (TNBC) typically receive neoadjuvant systemic therapy (NAST). However, the response of TNBC to NAST is varied and a prognostic marker is still lacking. Since angiogenesis plays an important role in cancer progression, the quantification of changes in the tumor vasculature during treatment has the potential to provide useful information for characterizing the treatment response. We have recently developed a method to quantify changes in tumor-associated vasculature through a novel analysis of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). In this study, we investigated its ability to monitor the response of TNBC to NAST. Methods: Dynamic contrast-enhanced (DCE) MRI was acquired in TNBC patients (N = 50; 25 pathological complete response (pCR), 25 non-pCR) before, after 2 and 4 cycles of Adriamycin/Cyclophosphamide (A/C), as part of the ARTEMIS trial (NCT02276433). Using DCE-MRI, we identified the tumor-associated vessels via a breast vasculature analysis. Specifically, the difference between pre- and post-contrast DCE-MRI images was enhanced by a histogram-based intensity transfer function. The 3D vasculature in the breast was then segmented by applying a Hessian filter. Given the vasculature segmented by the algorithms and the tumor masks segmented by radiologists, a lowest-cost tracking algorithm was used to automatically detect the vessels that are most likely to contact the tumor. These vessels are defined as tumor-associated vessels (TAV). The number of TAVs per patient was tabulated and the Wilcoxon rank sum test compared the TAV values before (V1), after 2 cycles (V2), and after 4 cycles of NAST (V3). Additionally, we compared the percent change of TAV from V1 to V3 between the pCR patients and non-pCR patients. Statistical significance was defined as P &amp;lt; 0.05. Results: A significant decrease in the number of TAVs was observed during the NAST. In particular, the number of TAVs has a median (interquartile range) of 15 (7 - 24), 7 (3 - 14), and 2 (0 - 8) at V1, V2, V3, respectively, which are (pair-wise) significantly different (P &amp;lt; 0.01). Moreover, pCR patients showed a significantly greater decrease in the number of TAVs as compared to non-pCR patients. The percent changes in the number of TAVs from V1 to V3 have a median (range) of -88.89% (-100.00% - -60.00%) and -66.67% (-87.85% - -40.88%) in the pCR and non-pCR patients, respectively (P &amp;lt; 0.05). Conclusion: These preliminary results demonstrate that tumor-associated vasculature may be a valuable imaging biomarker for monitoring the response of TNBC to NAST. Ongoing efforts include additional investigation of the TAVs beyond their number, as well as applying the analysis to more patients. Citation Format: Chengyue Wu, Casey E. Stowers, Zhan Xu, Ernesto A. B. F. Lima, Clinton Yam, Jong Bum Son, Jingfei Ma, Gaiane M. Rauch, Thomas E. Yankeelov. Quantification of tumor-associated vasculature as an imaging biomarker for monitoring the response of triple-negative breast cancer to neoadjuvant chemotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5569.

Abstract 1521: Unlocking the therapeutic index of highly potent site-specific ADCs in solid tumors

Abstract CD276 (B7-H3) is a transmembrane protein of the B7 family, which includes many prominent immune checkpoint targets, and is expected to play a role in T-cell stimulation, tumor-infiltration, as well as other tumorigenic functions with poor prognosis when upregulated. CD276 is highly expressed in solid tumors, cancer initiating stem cells, and tumor associated vasculature with minimal to no expression in healthy tissue. This promising expression profile has piqued interest for various targeted therapeutic modalities, such as antibody-drug conjugates (ADCs). Developing effective ADCs for solid tumors remains a challenge due to inefficient conjugation technologies which are either non-homogeneous, unstable, or do not position payloads at optimal sites. BrickADCs minimize hydrophobicity, optimize payload release kinetics, and tune biophysical properties to improve efficacy, safety, and bioavailability, resulting in improved therapeutic indexes. Potent payloads such as pyrrolobenzodiazepines have been underutilized due to toxicity, preventing their widespread use. BrickBio’s unique bioconjugation methodology enables precise (site-specific), flexible (unfettered site accessibility and chemistry), efficient, and scalable generation of ADCs which overcome these limitations. In this work, UAAs (unnatural amino acids) were incorporated into full-length anti-CD276 antibodies and conjugated with optimized pyrrolobenzodiazepine (PBD) payloads. BrickADCs leverage a proprietary Site-Select Panel to find optimal sites that shield the toxicity of highly potent, hydrophobic PBD payloads while maintaining their high activity to generate anti-CD276-UAA-PBD ADCs. The highest performing BrickADCs, developed by a rapid screen of PBD payloads conjugated to different sites in the antibody backbone, exhibit high therapeutic efficacy in a variety of CD276 positive solid tumor cell lines (eg. colon, ovarian, lung, sarcoma, etc) and in mouse xenograft models with a 3x differential in complete response. The tuned hydrophobicity and release kinetics have resulted in ADCs that have an increased therapeutic index, particularly due to their improved safety profile compared to cysteine conjugation.The BrickADC platform enables an increased therapeutic window for potent payloads across solid tumors, opening the floodgates for further development of ADCs which leverage payloads that have been plagued by poor therapeutic index. Continued work is underway for the CD276 pipeline candidates as they progress towards IND, as well as investigation into other targets and conjugate modalities. Citation Format: James S. Italia, Colby A. Souders, Sreedhar Reddy Suthe, Myer Hussain, Zhi Li, John Boyce, Audrey Warner, Abhishek Chatterjee. Unlocking the therapeutic index of highly potent site-specific ADCs in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1521.

Abstract 2501: A glioblastoma and blood-brain barrier in vitro model to assess the tumor microenvironment and temozolomide resistance

Abstract Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults with a dismal prognosis. Currently, Temozolomide (TMZ) and Bevacizumab are the main approved agents for primary and recurrent GBM, respectively. However, TMZ resistance is frequent and Bevacizumab does not improve overall survival. The heterogeneity of GBM and the presence of a highly selectively permeable blood-brain barrier (BBB) preclude the use of chemotherapeutics approved for other malignancies. Meanwhile, new therapy development is limited in part because most pre-clinical models do not recapitulate the key features of GBM that are relevant to therapy resistance and drug delivery. This is particularly important when validating immunotherapies such as engineered T cell therapy, which has shown some promise in the clinics. We therefore developed an in vitro 3D model that recapitulates the major features of the GBM tumor microenvironment (TME) that are involved in therapy resistance, including the dense tumor core and infiltrating invasive front observed in clinical tumors, surrounded by a perfusable BBB with a physiological permeability. With this model, we investigate the angiogenic nature of GBM, and the role of tumor organization and the TME in TMZ resistance and immunosuppression. We find that TMZ sensitivity decreases as complexity of tumor cell 3D organization increases, and identify pathways potentially associated with TMZ resistance and immunosuppression using proteomics and genomics approaches. Additionally, we find that tumor-associated vasculature has a higher permeability due to VEGFA from GBM tumors, which leads to the downregulation of endothelial junction proteins. Using an IL13Ra2-targetting chimeric antigen receptor (CAR) T cell as a model for engineered T cell therapy, we find that tumor-associated vasculature acts as a physical barrier to CAR-T cell extravasation, significantly impeding CAR-T cell efficacy. We then use the model to screen a CAR library to identify novel CAR combinations that can improve CAR-T cell extravasation and cytotoxicity in GBM. Citation Format: Maxine S. Lam, Joey Aw, Damien Tan, Ragavi Vijayakumar, Khloe S. Gordon, Michael E. Birnbaum, Radoslaw M. Sobota, Giulia Adriani, Andrea Pavesi. A glioblastoma and blood-brain barrier in vitro model to assess the tumor microenvironment and temozolomide resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2501.

Abstract 3511: Melanoma-shed, lymph-borne CSPG4 conditions the pre-metastatic lymph node niche

Abstract Tissues are primed for metastasis prior to the arrival of tumor cells. Primary tumors drive this transformation by shedding tumor-derived factors (TDFs), including extracellular vesicles and secreted/shed proteins (TSPs) into tumor-associated blood and lymphatic vasculature. Despite being the most common site of metastasis across solid tumor types, we know very little about how the lymph node (LN) is primed for metastasis by TDFs. Here, we test the hypothesis that lymph-borne TDFs are sufficient to initiate the formation of the pre-metastatic LN niche. Using a murine melanoma model (YUMM1.7), we demonstrate primary melanomas initiate remodeling in their draining LNs that enhances metastatic outgrowth relative to contralateral, non-draining and naïve LN controls. Furthermore, we have demonstrated that TDFs isolated from YUMM1.7-conditioned media uniquely activate this pro-metastatic LN phenotype rather than factors derived from benign dermis. The focus of this field has been on the role tumor-derived extracellular vesicles play in establishing this pre-metastatic niche, however our data further demonstrates TSPs are also sufficient to activate this niche. To identify candidate TDFs in vivo that have the potential to initiate LN transformation, we adopted a novel chemical biology strategy to ubiquitously tag TDFs in vivo using non-canonical amino acids and engineered expression of mutant tRNA synthetases in YUMM1.7 melanoma cells. Using this approach, we identified several TDFs by mass spectrometry present in pre-metastatic LNs, and identified one candidate (CSPG4; Melanoma-Associated Chondroitin Sulfate Proteoglycan) as a potential mediator of pre-metastatic niche development. Shed CSPG4 is found within TSPs isolated from media conditioned by YUMM1.7, but not benign dermal, cells. We generated stable shRNA-expressing YUMM1.7 cell lines for efficient knockdown of CSPG4, and found that loss of CSPG4 confers loss of TSP-dependent pre-metastatic niche development in the draining LN. Furthermore, using an enzyme specific to chondroitins (the main glycan decorating CSPG4), we demonstrate that loss of chondroitin glycosylation of CSPG4 is sufficient to attenuate metastasis, even with the core protein present. We find that fibroblastic reticular cells in the draining LN respond to TSPs by becoming more myofibroblast like, resembling cancer-associated fibroblasts that provide direct trophic support to tumor cells. This phenotypic switch is partially rescued in the absence of CSPG4. Our results collectively indicate TSPs prime the LN for metastasis and we have identified melanoma-shed CSPG4 as a driver of pre-metastatic niche development, potentially through its influence on the LN-resident fibroblasts. Citation Format: Haley du Bois, Annie L. Chen, Katherine S. Ventre, Maria S. Steele, Triantafyllia Karakousi, Amanda W. Lund. Melanoma-shed, lymph-borne CSPG4 conditions the pre-metastatic lymph node niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3511.