Unique Tumor Microenvironment Research Articles

A precision intelligent nanomissile for inhibiting tumor metastasis, boosting energy deprivation and immunotherapy

The epithelial-mesenchymal transition (EMT), tumor stroma and local metabolic alterations cooperate to establish a unique tumor microenvironment (TME) that fosters tumor progression and metastasis. To tackle this challenge, a precision intelligent nanomissile named HA@AT-Pd has been designed for dual-pronged cancer-associated fibroblast (CAF) transformation and tumor cell elimination. It is observed that HA@AT-Pd inhibits the production of cancer stem cells (CSCs) by blocking the TGF-β/Smad signaling pathway-mediated EMT and reversing activated CAFs to quiescence. Notably, HA@AT-Pd induces energy depletion in breast cancer cells through simultaneously suppressing cellular oxidative phosphorylation and glycolysis. The inhibition of glycolysis results in reduced lactic acid production, thereby converting an immunosuppressive TME into an immune-activating environment. Furthermore, the photothermal effect generated by HA@AT-Pd evokes immunogenic cell death, which can further enhance the anti-tumor immune response. Overall, this multifunctional combination strategy unveils potential therapeutic avenues to inhibit tumor progression and metastasis.

A single-cell perspective on immunotherapy for pancreatic cancer: from microenvironment analysis to therapeutic strategy innovation.

Pancreatic cancer remains one of the most lethal malignancies, with conventional treatment options providing limited efficacy. Recent advancements in immunotherapy have offered new hope, yet the unique tumor microenvironment (TME) of pancreatic cancer poses significant challenges to its successful application. This review explores the transformative impact of single-cell technology on the understanding and treatment of pancreatic cancer. By enabling high-resolution analysis of cellular heterogeneity within the TME, single-cell approaches have elucidated the complex interplay between various immune and tumor cell populations. These insights have led to the identification of predictive biomarkers and the development of innovative, personalized immunotherapeutic strategies. The review discusses the role of single-cell technology in dissecting the intricate immune landscape of pancreatic cancer, highlighting the discovery of T cell exhaustion profiles and macrophage polarization states that influence treatment response. Moreover, it outlines the potential of single-cell data in guiding the selection of immunotherapy drugs and optimizing treatment plans. The review also addresses the challenges and prospects of translating these single-cell-based innovations into clinical practice, emphasizing the need for interdisciplinary research and the integration of artificial intelligence to overcome current limitations. Ultimately, the review underscores the promise of single-cell technology in driving therapeutic strategy innovation and improving patient outcomes in the battle against pancreatic cancer.

First reported advanced pancreatic cancer with hyperprogression treated with PD-1 blockade combined with chemotherapy: a case report and literature review.

Pancreatic cancer is among the most immune-resistant tumor types due to its unique tumor microenvironment and low cancer immunogenicity. Single-agent immune modulators have thus far proven clinically ineffective. However, a growing body of evidence suggests that combination of these modulators with other strategies could unlock the potential of immunotherapy in pancreatic cancer. Herein, we describe the case of a 59-year-old male with metastatic pancreatic ductal adenocarcinoma, referred to our center to receive immunotherapy (serplulimab, a novel anti-PD-1 antibody) combined with chemotherapy (gemcitabine/nab-paclitaxel). During the initial three treatment cycles, the patient was assessed as having stable disease (SD) according to RECIST 1.1 criteria. However, following two additional cycles of combination therapy, the primary tumor mass increased from 4.9cm to 13.2cm, accompanied by the development of new lung lesions, ascites, and pelvic metastases. He succumbed to respiratory failure one month later. Retrospective analysis revealed that the patient had MDM4 amplification, identified as a high-risk factor for hyperprogressive disease (HPD). To our knowledge, this is the first reported case of HPD in pancreatic cancer with multiple metastases treated using combination therapy. We investigated the potential mechanisms and reviewed the latest literature on predictive factors for HPD. These findings suggest that while chemotherapy combined with immunotherapy may hold promise for treating pancreatic cancer, it is imperative to identify and closely monitor patients with high-risk factors for HPD when using immunotherapy.

PD-1 inhibitor sintilimab treated patients with metastatic triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly challenging subtype due to a unique tumor microenvironment. Several evidence (IMpassion130 trial and KEYNOTE-355 trial) supported the therapeutic effect of the immune checkpoint inhibitor in TNBC. However, the efficacy and safety of the PD-1 inhibitor sintilimab in breast cancer (BC) has not been well-investigated. So the real-world data on sintilimab-treated patients with metastatic BC were collected and analyzed in this study. The patients were eligible according to the requirements included: ages between 18years and 75years; recurrent or metastatic TNBC; measurable disease based on RECIST v1.1; no limitation on the prior systemic treatments; and ECOG performance status of 0-1. Patients received sintilimab 200mg intravenously every 3 weeks until unacceptable toxicity or disease progression. From 1 June 2019 to 1 October 2022, 40 female patients (median age, 55.5years) with metastatic TNBC (mTNBC) were enrolled into the study. The median prior lines of systemic therapy for mTNBC was three (range, 1-8), with 60% of cases receiving at least three lines of therapy for metastatic disease. The visceral or brain metastasis was detected in 40.4% or 9.6% of patients, respectively. The median duration of response was 2.8months (range, 0.7-21.0), and the median number of sintilimab doses administered was 4 (range, 1-30). The ORR and DCR were 22.5% and 72.5%, separately. The median PFS was 3.5 months (range, 1.4-21.0), with a 6-month PFS rate of 15.0% (6/40). The median OS was 52.5months (range, 9.0-247.0) as of data cut-off. Common adverse effects were acceptable, and fatigue, skin rash, and pruritus were the frequent toxicity observed. Two cases of grade 3 curable adverse events were considered to be treatment-related. PD-L1-positive tumor was found in 40% cases (4/10) of mTNBC. Although statistical difference was not reached, the trend was obvious. Patients with PD-L1-positive tumor gained better treatment response, while the TMB-high carrier received more benefits of PFS and OS. In our study, preliminary evidence provided the anticancer activity and acceptable adverse effects of sintilimab administered every 3 weeks to pretreated patients with mTNBC. Sintilimab showed its efficacy and safety of immunotherapy for patients with advanced TNBC.

Identification of Necroptosis-related Molecular Subtypes and Construction of Necroptosis-related Gene Signature for Glioblastoma Multiforme.

Necroptosis is a highly regulated and genetically controlled process, and therefore, attention has been paid to the exact effects of this disorder on a variety of diseases, including cancer. An in-depth understanding of the key regulatory factors and molecular events that trigger necroptosis can not only identify patients at risk of cancer development but can also help to develop new treatment strategies. This study aimed to increase understanding of the complex role of necroptosis in glioblastoma multiforme (GBM) and provide a new perspective and reference for accurate prediction of clinical outcomes and gene-targeted therapy in patients with GBM. The objective of this study was to analyze the gene expression profile of necroptosis regulatory factors in glioblastoma multiforme (GBM) and establish a necroptosis regulatory factor-based GBM classification and prognostic gene signature to recognize the multifaceted impact of necroptosis on GBM. The necroptosis score of the glioblastoma multiforme (GBM) sample in TCGA was calculated by ssGSEA, and the correlation between each gene and the necroptosis score was calculated. Based on necroptosis score-related genes, unsupervised consensus clustering was employed to classify patients. The prognosis, tumor microenvironment (TME), genomic changes, biological signal pathways and gene expression differences among clusters were analyzed. The gene signature of GBM was constructed by Cox and LASSO regression analysis of differentially expressed genes (DEGs). Based on 34 necroptosis score-related genes, GBM was divided into two clusters with different overall survival (OS) and TME. A necroptosis-related gene signature (NRGS) containing 8 genes was developed, which could stratify the risk of GBM in both the training set and verification set and had good prognostic value. NRGS and age were both independent prognostic indicators of GBM, and a nomogram developed by the integration of both of them showed a better predictive effect than traditional clinical features. In this study, patients from public data sets were divided into two clusters and the unique TME and molecular characteristics of each cluster were described. Furthermore, an NRGS was constructed to effectively and independently predict the survival outcome of GBM, which provides some insights for the implementation of personalized precision medicine in clinical practice.

Malignancy of Malignant Ascites: A Comprehensive Review of Interplay between Biochemical Variables, Tumor Microenvironment and Growth Factors.

Malignant ascites, a buildup of fluid in the abdominal cavity, is a serious consequence of many malignancies. This review aims to comprehend the biochemical makeup of malignant ascites, such as pH, cholesterol, protein, etc., which is crucial to developing therapeutics with better treatment outcomes and hence correlate with corresponding prognostic value. The unique tumour microenvironment exhibited by malignant ascites and the crosstalk between inflammatory cells, cytokines and chemokines, interactions between tumour and non-tumour cell types, activation of vital cell signalling pathways within the TME for VEGF-regulated sustained angiogenesis, cancer progression and metastasis is highlighted. This review addresses the need to develop comprehensive assay platforms to identify various biochemical aspects of ascites, to discover the interactions of the tumour microenvironment and to study VEGF-regulated permeability that can expedite early diagnosis and progression of ascites.

Higher Microbial Abundance and Diversity in Bronchus-Associated Lymphoid Tissue (BALT) Lymphomas than in Non-Cancerous Lung Tissues.

It is well known that the majority of the extranodal marginal zone lymphomas of mucosa-associated lymphoid tissues (MALT lymphomas) are associated with microbiota, e.g., gastric MALT lymphoma with Helicobacter pylori. In general, they are very sensitive to low-dose radiotherapy and chemotherapeutic agents. The microbiota profile is not clearly elucidated in bronchus-associated lymphoid tissue (BALT) lymphoma, a rare type of MALT lymphoma in the lung. Thus, this study aimed to clarify the intratumor microbiome in BALT lymphoma using the third-generation NGS method. DNAs were extracted from 12 formalin-fixed paraffin-embedded (FFPE) tumor tissues obtained from BALT lymphoma patients diagnosed between 1990 and 2016. 16S rRNA gene was amplified by polymerase chain reaction. Amplicons were sequenced using a Nanopore platform. Next-generation sequencing analysis was performed to assess microbial profiles. For comparison, FFPE specimens from nine non-cancerous lung tissues were also analyzed. Specific bacterial families including Burkholderiaceae, Bacillaceae, and Microbacteriaceae were associated with BALT lymphoma by a linear discriminant analysis effect size approach. Although the number of specimens was limited, BALT lymphomas exhibited significantly higher microbial abundance and diversity with distinct microbial composition patterns and correlation networks than non-cancerous lung tissues. This study provides the first insight into intratumor microbiome in BALT lymphoma using the third-generation NGS method. A distinct microbial composition suggests the presence of a unique tumor microenvironment of BALT lymphoma.

Unlocking the tumor-immune microenvironment in osteosarcoma: insights into the immune landscape and mechanisms.

Osteosarcoma has a unique tumor microenvironment (TME), which is characterized as a complex microenvironment comprising of bone cells, immune cells, stromal cells, and heterogeneous vascular structures. These elements are intricately embedded in a mineralized extracellular matrix, setting it apart from other primary TMEs. In a state of normal physiological function, these cell types collaborate in a coordinated manner to maintain the homeostasis of the bone and hematopoietic systems. However, in the pathological condition, i.e., neoplastic malignancies, the tumor-immune microenvironment (TIME) has been shown to promote cancer cells proliferation, migration, apoptosis and drug resistance, as well as immune escape. The intricate and dynamic system of the TIME in osteosarcoma involves crucial roles played by various infiltrating cells, the complement system, and exosomes. This complexity is closely associated with tumor cells evading immune surveillance, experiencing uncontrolled proliferation, and facilitating metastasis. In this review, we elucidate the intricate interplay between diverse cell populations in the osteosarcoma TIME, each contributing uniquely to tumor progression. From chondroblastic and osteoblastic osteosarcoma cells to osteoclasts, stromal cells, and various myeloid and lymphoid cell subsets, the comprehensive single-cell analysis provides a detailed roadmap of the complex osteosarcoma ecosystem. Furthermore, we summarize the mutations, epigenetic mechanisms, and extracellular vesicles that dictate the immunologic landscape and modulate the TIME of osteosarcoma. The perspectives of the clinical implementation of immunotherapy and therapeutic approaches for targeting immune cells are also intensively discussed.

Abstract C010: Single-cell profiling unleashes KRAS-driven redrafting of the tumor microenvironment before cancer onset

Abstract Pancreatic cancer is a highly lethal and aggressive disease, commonly characterized by a unique tumor microenvironment, where mutant KRAS signaling plays a pivotal role in its pathophysiology. To deepen our understanding of KRAS-dependent signaling in earliest tumor development, oncogenic KRAS (KRASG12D) expression was induced in human stem cell-derived pancreatic ductal-like organoids (PDLOs), followed by time-resolved single-cell RNA and bulk ATAC sequencing. This unique precancerous state model facilitated a comprehensive characterization of early oncogenic events at cellular resolution. Furthermore, a machine learning approach was applied to accurately predict KRAS-dependent cell identities. Within seven days, mutated KRAS triggers distinct gene signature programs related to extracellular matrix remodeling and inflammation. Although derived from dysplastic yet non-cancerous states, these gene signatures negatively correlated with overall survival in PDAC (pancreatic ductal adenocarcinoma) patients. The KRAS-dependent signatures retrieved with the machine learning approach allowed the grouping of 24 published PDAC cases, showing differences in niche composition and communication patterns. Various co-culture systems demonstrated that KRASG12D-expressing PDLOs could activate pancreatic stellate cells, inducing a cancer- associated fibroblast (CAF)-like state. Simultaneously, these KRAS-activated organoids were protected from T cell infiltration in vitro. Furthermore, in silico approaches were employed to reconstruct a virtual pancreatic cancer space, demonstrating that our PDLOs transcriptionally bridge healthy and malignant ductal states. This also revealed potential KRAS-driven pathways mediating CAF activation and T cell shielding preceding cancer onset. In summary, our human PDLO model enables us to explore the transition from normal to cancerous states, addressing gaps in our understanding of tumor development and tumor niche preparation. Citation Format: Chantal Allgoewer, Markus Breunig, Meike Hohwieler, Medhanie Mulaw, Alexander Kleger. Single-cell profiling unleashes KRAS-driven redrafting of the tumor microenvironment before cancer onset [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C010.

Abstract A055: Multiomics profiling reveals druggable tumor-stroma interactions in ATM-deficient pancreatic cancer

Abstract The complex biology of pancreatic ductal adenocarcinoma (PDAC) remains a significant challenge and limits overcoming its dismal prognosis. The tumor microenvironment (TME), shaped by dynamic interactions between cancer-associated fibroblasts (CAFs), immune and tumor cells, plays a crucial role in PDAC pathogenesis. Uncharted field lies in the impact of specific gene mutations within the tumor epithelium on orchestrating distinct oncogenic TME patterns. Our investigation into homologous recombination deficiency (HRD), caused by mutations in genes like ATM, reveals its role in promoting PDAC aggressiveness and triggering a pronounced desmoplastic reaction, suggesting a unique TME programing. Through single-cell-resolved and multiomics analyses of murine ATM and/or P53-depleted PDACs, and validation in human cases, we unveil transcriptional, translational, and secretory regulatory events, shed light on the intricate interplay between tumor genetics and PDAC microenvironment and pave the way for the elaboration of novel personalized therapies. Murine tumor analysis using single-nucleus multiomics RNA and ATAC sequencing, alongside histological validation in human tissues, revealed specific enrichment of myCAFs in ATM-deficient tumors and reduced populations of T cells and macrophages. Molecular signatures and cell-cell communication inference uncovered ATM-specific interactions within the TME, highlighting TGF-β signaling as pivotal in mediating dialog between CAFs and an aggressive EMT-like tumor cell subset prevalent in ATM-depleted tumors. Additionally, distinct communication patterns were observed between CAFs and immune cells, associated with specific epigenetic and molecular profiles. Mechanistically, transcriptomics, secretomics and proteomics investigations confirmed increased TGFβ release by ATM-deficient PDAC cells and identified a reactive oxygen species (ROS)-mediated mechanism affecting tumor cell behavior and interactions with CAFs. Consistently, a combinatorial therapy targeting TGF-β-mediated tumor-stroma dialog reversed cancer-promoting TME remodeling and exacerbated FOLFIRINOX cytotoxic effects, in vivo, exclusively in ATM-deficient HRD PDACs. Dissecting the biology of ATM-deficient HRD malignant cells, our study unveils their pivotal role in orchestrating oncogenic communication networks with CAFs and immune cells, along with specific tumor promoting stromal reprogramming. Our findings paint a comprehensive picture of how distinct tumor-stroma interactions reshape the TME, steering it towards a cancer-promoting outcome and shed light on the potential to exploit genotype-specific vulnerabilities via tailored tumor-stroma interference strategies. Citation Format: Elodie Roger, Hannah M. Mummey, Eleni Zimmer, Julia Ragg, Yuna Lee, Kyle J. Gaulton, Alica K. Beutel, Christopher J. Halbrook, Lukas Perkhofer, Johann Gout, Alexander Kleger. Multiomics profiling reveals druggable tumor-stroma interactions in ATM-deficient pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A055.

Environment-responsive dopamine nanoplatform for tumor synergistic therapy

Nanoparticle-based photothermal therapy (PTT) has emerged as a promising approach in tumor treatment due to its high selectivity and low invasiveness. However, the penetration of near-infrared light (NIR) is limited, leading it fails to induce damage to the deep-seated tumor cells within the tumor tissue. Additionally, inefficient uptake of photothermal nanoparticles by tumor cells results in suboptimal outcomes for PTT. In this study, we utilized the adhesive properties of photothermal material, polydopamine (PDA), which can successfully load the photosensitizer indocyanine green (ICG) and chemotherapeutic drug doxorubicin (DOX) to achieve photothermal and chemotherapy synergy treatment (PDA/DOX&ICG), aiming to compensate the defects of single tumor treatment. To extending the blood circulation time of PDA/DOX&ICG nanoparticles, evading clearance by the body immune system and achieving targeted delivery to tumor tissues, a protective envelopment was created using erythrocyte membranes modified with folate acid (FA-EM). After reaching the tumor tissue, the obtained FA-EM@PDA/DOX&ICG nanoparticles can specific bind with folate acid receptors on the surface of tumor cells, which can improve the uptake behavior of FA-EM@PDA/DOX&ICG nanoparticles by tumor cells, and leading to the release of loaded DOX and ICG in response to the unique tumor microenvironment. ICG, as a typical photosensitizer, significantly enhances the photothermal conversion performance of FA-EM@PDA/DOX&ICG nanoparticles, thus inducing tumor cells damage. In vitro and in vivo experimental results demonstrated that the coordinated NIR treatment with FA-EM@PDA/DOX&ICG not only effectively inhibits tumor growth, but also exhibits superior biocompatibility, effectively mitigating DOX-induced tissue damage.

Tertiary Lymphoid Structures in Brain Metastases of Lung Cancer: Prognostic Significance and Correlation With Clinical Outcomes.

The prognosis of patients with brain metastases (BMs) originating from lung cancer remains poor, despite advancements in treatment strategies. The role of tertiary lymphoid structures (TLSs) within the tumor immune microenvironment of BMs has not been extensively explored. This study utilized patient-derived clinical samples from 17 patients with histologically confirmed BMs of lung cancer, undergoing surgical resection. Immunohistochemistry was employed to analyze the presence and characteristics of TLS and tumor-infiltrating lymphocytes (TILs) within BM tissues, correlating these with clinical outcomes. TLSs, albeit in their immature form, were identified within BM tissues, distinguishing them from their mature counterparts in primary lung cancer tissues. A significant correlation between TLS density (but not TIL density) and improved postoperative survival was observed, underscoring the potential of TLS density as an independent prognostic marker. Furthermore, TLS density did not correlate with the Graded Prognostic Assessment (GPA) index, suggesting its unique prognostic value beyond conventional predictors. Our findings reveal the presence of TLSs in lung cancer-derived BMs and highlight their prognostic significance, independent of the GPA index. The identification of TLS within the unique central nervous system tumor microenvironment offers new insights into the immune landscape of BMs and suggests potential avenues for immunotherapeutic interventions targeting these structures to improve patient outcomes.

Development and efficacy evaluation of nanoliposomes targeting CAFs-LCSCs communication for hepatocellular carcinoma treatment

Hepatocellular carcinoma (HCC) is a prevalent global malignancy, presenting significant challenges in developing safe and effective treatments to prevent recurrence. This is mainly attributed to the inability of traditional treatments to eliminate liver cancer stem cells (LCSCs). LCSCs exhibit resistance to standard therapies, such as radiotherapy and chemotherapy, playing a noticeable role in drug resistance, metastasis, and recurrence of HCC. Their “stemness” is maintained by cancer-associated fibroblasts (CAFs), especially dense CAFs that form a barrier, hindering drug delivery. These two components interact to drive the progression of HCC. Regarding this unique tumor microenvironment, a novel nanoliposome (CAP@CD133-D/X-Lip) was developed. This nanoliposome could initially target CAFs by leveraging fibroblast activating protein (FAP) overexpression on their surface, activating CAP-degrading peptides upon reaching CAFs. Subsequent size reduction and exposure of CD133 aptamers enable secondary targeting of LCSCs, leading to the release of the antitumor drug doxorubicin hydrochloride (DOX) and the LCSCs’ inhibitor XAV-939 in deep tumor regions. The efficacy of CAP@CD133-D/X-Lip was validated in vitro and in vivo, demonstrating its ability to target therapeutic drugs and disrupt the ’CAFs-LCSCs’ communication, thereby slowing HCC progression and enhancing treatment outcomes. This approach holds significant promise for the clinical management of HCC.

DIPG-55. DISSECTING THE ECOSYSTEM OF PEDIATRIC DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Pediatric gliomas are heterogeneous tumors encompassing high grade (pHGG) and low grade (pLGG) subtypes. For tumors not amenable to surgery, in particular high grade H3K27 mutant diffuse midline glioma (DMG), no curative options exist. DMG is characterized by 1) extensive heterogeneity of tumor cells that can exist in different interchangeable cellular states mimicking normal development and 2) extensive heterogeneity of the tumor microenvironment (TME). A better understanding of the DMG ecosystem is needed for therapeutic target identification. METHODS Publicly available single cell RNA seq data of DMG (n=17, Jessa et al. Nat. Genet. 2022), high grade pediatric glioblastoma (GBM, n=16, scPCA) and low grade tumors (ganglioglioma, n=5; pilocytic astrocytoma, n=14, scPCA) were analyzed to define key molecular characteristics and differences between these pediatric brain tumors. Doublet cells were removed from each dataset and tumor and TME cells were discriminated based on marker gene expression and inferred CNVs. RESULTS In DMG, glioma cellular states and TME heterogeneity differed depending on the tumor location. DMG arising in the pons harbored a bigger fraction of astrocyte-like glioma cells whereas oligodendrocyte-like cells were more abundant in thalamic tumors. In line with this observation, Liana cellular communication analysis inferred more intratumoral (autocrine) signaling involving oligodendrocyte-like glioma cells in thalamic tumors (e.g. NCAM1-PTPRZ1, NLGN4X-NRXN1, NTN1-DSCAM) than in pons tumors. Notably, the TME of thalamic tumors contained a bigger fraction of immunotolerant macrophages expressing CD163 or VSIG4. In contrast, pons tumors harbored a unique cluster of VEGFA expressing astrocytes, not found in GBM or pLGG. Liana analysis inferred COPA/APP-CD74 interaction between VEGFA+ astrocytes and CD163/VSIG4+ macrophages and microglia, potentially contributing to an immunotolerant TME in pons DMG. CONCLUSIONS In DMG, patterns of tumor and TME composition associated with tumor location were uncovered. Furthermore, unique TME celltypes could contribute to the particularly aggressive nature of these tumors.

Biomimetic Hydrogel-Mediated Mechano-Immunometabolic Therapy for Inhibition of ccRCC Recurrence After Surgery.

The unique physical tumor microenvironment (TME) and aberrant immune metabolic status are two obstacles that must be overcome in cancer immunotherapy to improve clinical outcomes. Here, an in situ mechano-immunometabolic therapy involving the injection of a biomimetic hydrogel is presented with sequential release of the anti-fibrotic agent pirfenidone, which softens the stiff extracellular matrix, and small interfering RNA IDO1, which disrupts kynurenine-mediated immunosuppressive metabolic pathways, together with the multi-kinase inhibitor sorafenib, which induces immunogenic cell death. This combination synergistically augmented tumor immunogenicity and induced anti-tumor immunity. In mouse models of clear cell renal cell carcinoma, a single-dose peritumoral injection of a biomimetic hydrogel facilitated the perioperative TME toward a more immunostimulatory landscape, which prevented tumor relapse post-surgery and prolonged mouse survival. Additionally, the systemic anti-tumor surveillance effect induced by local treatment decreased lung metastasis by inhibiting epithelial-mesenchymal transition conversion. The versatile localized mechano-immunometabolic therapy can serve as a universal strategy for conferring efficient tumoricidal immunity in "cold" tumor postoperative interventions.

Immunotherapy outcomes and profile in lung cancer brain metastases.

8646 Background: Brain metastasis (BM) in NSCLCs have a unique tumor micro-environment (TME) characterized by distinct immune-constituents and the presence of blood-brain barrier, differentiating them from primary NSCLC tumors (PT). Despite the high incidence of BM, earlier trials indicated that less than 15% of BM patients benefit from immune checkpoint inhibitors (ICIs). We characterized the TME of BM to potentially identify a subset of patients who may respond favorably to ICIs. Methods: 36,726 NSCLC samples were analyzed by NGS of DNA (592-gene) and RNA (whole transcriptome) at Caris Life Sciences (Phoenix, AZ). TMB-high was defined as >=10mt/MB, PD-L1 was tested by IHC (22c3), and TME by RNA expression (quanTIseq). Real-world overall survival was obtained from insurance claims data and calculated from treatment start on six common ICIs to last contact (IO-OS), while time-on-treatment (TOT) was from first to last of treatment. Hazard ratios (HRs) were calculated using the Cox proportional hazards model and p values (log-rank test). Fisher’s exact tests and Mann-Whiney U were used and adjusted for multiple comparisons (q<0.05). Results: Samples were comprised of 63.5%, PT 4.9% BM, and 31.6% extracranial metastases. High TMB and loss of heterozygosity (LOH) rates were higher in BM vs. PT (55% vs. 35.7%) and (28.0% vs. 14.3%), q<0.05, respectively, while PD-L1+ rate was similar (53.3% BM vs. 55.6% PT). Significant mutational differences in BM vs. PT included TP53 (77.7% vs 65.1%), KEAP1 (19.4% vs. 12.1%), and STK11 (17.5% vs. 12.0%), q<0.05. Immune deconvolution demonstrated the abundance of neutrophils, Tregs, monocytes, and B cells was higher in PT vs. BM (fold-change: 1.35-1.73, q<0.05), while dendritic cells were higher in BM vs. PT (fold-change: 13, q<0.05). BM had significantly longer IO-OS (24.4 vs. 19.7 mo: HR = 0.83, 95% CI 0.76-0.90, p<0.05) and TOT (7.7 vs. 6.0 mo; HR = 0.83, 95% CI 0.77-0.89, p<0.05) compared to PT. PD-L1+ and TMB High were associated with longer IO-OS in BM vs. PT (HR = 0.71, 95% CI 0.63-0.81, p<0.05) and (HR = 0.83, 95% CI 0.74 – 0.93, p<0.05), respectively. By histology, squamous cell carcinoma (SC) was significantly more prevalent in PT compared to BM (25.9% vs. 8.5%, q<0.05). In adenocarcinoma (AD), BM was associated with longer IO-OS (HR = 0.78, 95% CI 0.70-0.87, p<0.05) compared to PT, while in SC, the survival association was not significant. The longer survival seen in AD was still significant after multivariate analysis with TP53, STK11 and KEAP1 (HR = 0.76, 95% CI 0.68-0.86, q<0.05). In BM, PD-L1+ and TMB High were associated with longer IO-OS in AD and may be considered first line treatment in this population, if asymptomatic. Conclusions: Historically BM have been associated with poor outcomes. We demonstrate that BM has higher TMB, increased infiltration of dendritic cells, and higher LOH than PT. After stratifying patients based on PD-L1 and TMB, patients with BM adenocarcinoma histology had improved post ICI survival.

Enrichment of glioblastoma tumor microenvironment in a GZMK+ T cell population.

2065 Background: Glioblastoma (GBM) bears a survival estimate below 10% at 5 years, despite standard chemoradiation treatment. Trials evaluating immune checkpoint blockade (ICB) in gliomas have been disappointing so far, dealing with the unique immunosuppressive tumor microenvironment (TME) of the brain. Nevertheless, we have clinical evidence of ICB efficacy for brain metastases (BrM) of immunosensitive tumors. Even if both GBMs and BrM share the same organ-specific microenvironment, it is clear that disease-specific immune characteristics instruct differential response to ICB. Methods: By using advanced deep learning algorithms to combine public and in-house-generated single-cell RNA sequencing profiles, we analyzed immune infiltrate characteristics of 602949 CD45+ cells from 76 GBM, 30 BrM from different types of extracranial tumors (13 NSCLC, 12 melanoma, 2 ovarian, 1 breast, 1 colorectal and 1 renal carcinoma) and 3 normal brain samples. The analysis was restricted to newly diagnosed GBM, excluding recurrent and pre-treated cases. CD8+ cells underwent segregation into clusters based on log2 normalized expression levels of CD3E and CD8A (with expression values greater than 0). The data integration utilized DESC, a deep learning model founded on autoencoders, and subsequent examination was conducted within the Scanpy framework. Cluster analysis was executed employing the Leiden algorithm. Differentially expressed genes (DEGs) were pinpointed across clusters using the Wilcoxon test, with False Discovery Rate (FDR) correction carried out through the Benjamini-Hochberg procedure. Results: From CD45+CD3+ cells, we isolated CD8+ T cells and identified 7 informative CD8+ T cells clusters. Based on DEGs, we could appreciate T cells clusters that were also recapitulated in other extracranial malignancies. The most abundant subset was composed of GZMK+ effector T cells (Cluster 0: GZMK, GZMM, CD44, KLRG1, IL7R, GZMA, CXCR3), followed by putative tumor-specific tissue-resident memory cells (Cluster 1: GZMB, LAG3, CTLA4, TIGIT, CXCL13, ITGAE, ENTPD1, PDCD1, CD27, HLA-DRB1), stem-cell memory-like T cells (Cluster 3: IL7R, CCR7, JUNB, KLF2, TCF7) and a less represented cluster characterized by high expression of genes belonging to the HSP family (Cluster 4: HSPA1, HSPH1, HSPE1, HSPD1, IFN). Interestingly, while the majority of the identified clusters was equally represented in the 3 different tissues, the GZMK+ effector T cells cluster (Cluster 0) was found to be more represented in GBM compared to BrM and normal brain tissue. Conclusions: A GZMK+ effector T cells subpopulation, specifically enriched in GBM, has been poorly characterized in its function and spatial localization. Our data highlight the importance of studying the composition of the immune infiltrate in GBM with multi-omics approaches in order to uncover deep mechanisms of resistance to ICB and eventually provide hints on potential therapeutic targets.

The role of the tumor microenvironment in papillary thyroid microcarcinoma nodal metastasis.

The genetic alterations currently identified in papillary thyroid microcarcinomas (PTMCs) are insufficient for distinguishing tumors with aggressive features. We aimed to identify candidate markers associated with lateral lymph node metastasis (LLNM, N1sb disease) in patients with PTMC using transcriptomic analysis. RNA sequencing was performed on 26 matched tumor and normal thyroid tissue samples (N0, n = 14; N1b, n = 12), followed by functional enrichment analyses of differentially expressed genes (DEGs). EcoTyper was used to explore the distinct tumor microenvironment (TME). We identified 631 DEGs (213 upregulated and 418 downregulated) between N1b and N0 PTMCs. The most significantly upregulated genes in N1b were associated with tumorigenesis, adhesion, migration, and invasion. DEGs were mainly enriched in the pathways of idiopathic pulmonary fibrosis, TME, wound healing, and inhibition of matrix metalloproteases. We predicted the activation of these pathways in N1b PTMCs. N1b PTMCs had a unique TME with abundant fibroblasts and epithelial cells, associated with an increased risk of disease progression. Fibroblast marker genes, including POSTN, MMP11, TNFAIP6,and FN1, and epithelial cell marker genes, including NOX4, MFAP2, TGFVBI,and TNC, were selected. POSTN and FN1, fibroblast cell-specific genes, and NOX4 and TNC, epithelial cell-specific genes, were promising biomarkers for predicting LLNM development and recurrence in patients with PTMC. We delineated the cellular ecotypes within the TME of patients with N1b PTMC and revealed potential markers for predicting LLNM and the prognosis of PTMC. These findings provide valuable insights into the contributions of cancer-associated fibroblasts and epithelial cells to PTMC progression and metastasis.

Cascade Self-Generation of Carbon Monoxide Triggered by Photoinduced Holes for Efficient Hypoxic Tumors Therapy.

It still remains challenging to design multifunctional therapeutic reagents for effective cancer therapy under a unique tumor microenvironment including insufficient endogenous H2O2 and O2, low pH, and a high concentration of glutathione (GSH). In this work, a CO-based phototherapeutic system triggered by photogenerated holes, which consisted of ionic liquid (IL), the CO prodrug Mn2(CO)10, and iridium(III) porphyrin (IrPor) modified carbonized ZIF-8-doped graphitic carbon nitride nanocomposite (IL/ZCN@Ir(CO)), was designed for cascade hypoxic tumors. Upon light irradiation, the photogenerated holes on IL/ZCN@Ir(CO) oxidize water into H2O2, which subsequently induces Mn2(CO)10 to release CO. Meanwhile, IrPor can convert H2O2 to hydroxyl radical (•OH) and subsequent singlet oxygen (1O2), which further triggers CO release. Moreover, the degraded MnO2 shows activity for glutathione (GSH) depletion and mimics peroxidase, leading to GSH reduction and •OH production in tumors. Thus, this strategy can in situ release high concentrations of CO and reactive oxygen species (ROS) and deplete GSH to efficiently induce cell apoptosis under hypoxic conditions, which has a high inhibiting effect on the growth of tumors, offering an attractive strategy to amplify CO and ROS generation to meet therapeutic requirements in cancer treatment.

Keratinocytes Present Staphylococcus aureus Enterotoxins and Promote Malignant and Nonmalignant T Cell Proliferation in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma is characterized by malignant T cells proliferating in a unique tumor microenvironment dominated by keratinocytes (KCs). Skin colonization and infection by Staphylococcus aureus are a common cause of morbidity and are suspected of fueling disease activity. In this study, we show that expression of HLA-DRs, high-affinity receptors for staphylococcal enterotoxins (SEs), by KCs correlates with IFN-γ expression in the tumor microenvironment. Importantly, IFN-γ induces HLA-DR, SE binding, and SE presentation by KCs to malignant T cells from patients with Sézary syndrome and malignant and nonmalignant T-cell lines derived from patients with Sézary syndrome and mycosis fungoides. Likewise, preincubation of KCs with supernatant from patient-derived SE-producing S aureus triggers proliferation in malignant T cells and cytokine release (including IL-2), when cultured with nonmalignant T cells. This is inhibited by pretreatment with engineered bacteriophage S aureus-specific endolysins. Furthermore, alteration in the HLA-DR-binding sites of SE type A and small interfering RNA-mediated knockdown of Jak3 and IL-2Rγ block induction of malignant T-cell proliferation. In conclusion, we show that upon exposure to patient-derived S aureus and SE, KCs stimulate IL-2Rγ/Jak3-dependent proliferation of malignant and nonmalignant T cells in an environment with nonmalignant T cells. These findings suggest that KCs in the tumor microenvironment play a key role in S aureus-mediated disease activity in cutaneous T-cell lymphoma.