Tumor Microenvironment Remodeling Research Articles

The involvement and application potential of exosomes in breast cancer immunotherapy

Breast cancer has a high incidence and a heightened propensity for metastasis. The absence of precise targets for effective intervention makes it imperative to devise enhanced treatment strategies. Exosomes, characterized by a lipid bilayer and ranging in size from 30 to 150 nm, can be actively released by various cells, including those in tumors. Exosomes derived from distinct subsets of immune cells have been shown to modulate the immune microenvironment within tumors and influence breast cancer progression. In addition, tumor-derived exosomes have been shown to contribute to breast cancer development and progression and may become a new target for breast cancer immunotherapy. Tumor immunotherapy has become an option for managing tumors, and exosomes have become therapeutic vectors that can be used for various pathological conditions. Edited exosomes can be used as nanoscale drug delivery systems for breast cancer therapy, contributing to the remodeling of immunosuppressive tumor microenvironments and influencing the efficacy of immunotherapy. This review discusses the regulatory role of exosomes from different cells in breast cancer and the latest applications of exosomes as nanoscale drug delivery systems and immunotherapeutic agents in breast cancer, showing the development prospects of exosomes in the clinical treatment of breast cancer.

Multiple Synergistic Effects of the Microglia Membrane-Bionic Nanoplatform on Mediate Tumor Microenvironment Remodeling to Amplify Glioblastoma Immunotherapy.

Glioblastoma (GBM) is a lethal brain tumor with high levels of malignancy. Most chemotherapy agents show serious systemic cytotoxicity and restricted delivery effectiveness due to the impediments of the blood-brain barrier (BBB). Immunotherapy has developed great potential for aggressive tumor treatments. Disappointingly, its efficacy against GBM is hindered by the immunosuppressive tumor microenvironment (TME) and BBB. Herein, a multiple synergistic immunotherapeutic strategy against GBM was developed based on the nanomaterial-biology interaction. We have demonstrated that this BM@MnP-BSA-aPD-1 can transverse the BBB and target the TME, resulting in amplified synergetic effects of metalloimmunotherapy and photothermal immunotherapy (PTT). The journey of this nanoformulation within the TME contributed to the activation of the stimulator of the interferon gene pathway, the initiation of the immunogenic cell death effect, and the inhibition of the programmed cell death-1/programmed cell death ligand 1 (PD-1/PD-L1) signaling axis. This nanomedicine revitalizes the immunosuppressive TME and evokes the cascade effect of antitumor immunity. Therefore, the combination of BM@MnP-BSA-aPD-1 and PTT without chemotherapeutics presents favorable benefits in anti-GBM immunotherapy and exhibits immense potential for clinical translational applications.

Intelligent nanovesicle for remodeling tumor microenvironment and circulating tumor chemoimmunotherapy amplification

Imperceptible examination and unideal treatment effect are still intractable difficulties for the clinical treatment of pancreatic ductal adenocarcinoma (PDAC). At present, despite 5-fluorouracil (5-FU), as a clinical first-line FOLFIRINOX chemo-drug, has achieved significant therapeutic effects. Nevertheless, these unavoidable factors such as low solubility, lack of biological specificity and easy to induce immunosuppressive surroundings formation, severely limit their treatment in PDAC. As an important source of energy for many tumor cells, tryptophan (Trp), is easily degraded to kynurenine (Kyn) by indolamine 2,3- dioxygenase 1 (IDO1), which activates the axis of Kyn-AHR to form special suppressive immune microenvironment that promotes tumor growth and metastasis. However, our research findings that 5-FU can induce effectively immunogenic cell death (ICD) to further treat tumor by activating immune systems, while the secretion of interferon-γ (IFN-γ) re-induce the Kyn-AHR axis activation, leading to poor treatment efficiency. Therefore, a metal matrix protease-2 (MMP-2) and endogenous GSH dual-responsive liposomal-based nanovesicle, co-loading with 5-FU (anti-cancer drug) and NLG919 (IDO1 inhibitor), was constructed (named as ENP919@5-FU). The multifunctional ENP919@5-FU can effectively reshape the tumor immunosuppression microenvironment to enhance the effect of chemoimmunotherapy, thereby effectively inhibiting cancer growth. Mechanistically, PDAC with high expression of MMP-2 will propel the as-prepared nanovesicle to dwell in tumor region via shedding PEG on the nanovesicle surface, effectively enhancing tumor uptake. Subsequently, the S-S bond containing nanovesicle was cut via high endogenous GSH, leading to the continued release of 5-FU and NLG919, thereby enabling circulating chemoimmunotherapy to effectively cause tumor ablation. Moreover, the combination of ENP919@5-FU and PD-L1 antibody (αPD-L1) showed a synergistic anti-tumor effect on the PDAC model with abdominal cavity metastasis. Collectively, ENP919@5-FU nanovesicle, as a PDAC treatment strategy, showed excellent antitumor efficacy by remodeling tumor microenvironment to circulate tumor chemoimmunotherapy amplification, which has promising potential in a precision medicine approach.

Phosphoglycerate mutase 1 promotes breast cancer progression through inducing immunosuppressive M2 macrophages.

Immunosuppressive tumor microenvironment (TME) contributes to tumor progression and causes major obstacles for cancer therapy. Phosphoglycerate mutase 1 (PGAM1) is a key enzyme involved in cancer metabolism while its role in remodeling TME remains unclear. In this study, we reported that PGAM1 suppression in breast cancer (BC) cells led to a decrease in M2 polarization, migration, and interleukin-10 (IL-10) production of macrophages. PGAM1 regulation on CCL2 expression was essential to macrophage recruitment, which further mediated by activating JAK-STAT pathway. Additionally, the CCL2/CCR2 axis was observed to participate in PGAM1-mediated immunosuppression via regulating PD-1 expression in macrophages. Combined targeting of PGAM1 and the CCL2/CCR2 axis led to a reduction in tumor growth in vivo. Furthermore, clinical validation in BC tissues indicated a positive correlation between PGAM1, CCL2 and macrophage infiltration. Our study provides novel insights into the induction of immunosuppressive TME by PGAM1 and propose a new strategy for combination therapies targeting PGAM1 and macrophages in BC.

NK cell transfer overcomes resistance to PD-(L)1 therapy in aged mice

BackgroundCancer is the leading cause of death among older adults. Although the integration of immunotherapy has revolutionized the therapeutic landscape of cancer, the complex interactions between age and immunotherapy efficacy remain incompletely defined. Here, we aimed to elucidate the relationship between aging and immunotherapy resistance.MethodsFlow cytometry was performed to evaluate the infiltration of immune cells in the tumor microenvironment (TME). In vivo T cell proliferation, cytotoxicity and migration assays were performed to evaluate the antitumor capacity of tumor antigen-specific CD8+ T cells in mice. Real-time quantitative PCR (qPCR) was used to investigate the expression of IFN-γ-associated gene and natural killer (NK)-associated chemokine. Adoptive NK cell transfer was adopted to evaluate the effects of NK cells from young mice in overcoming the immunotherapy resistance of aged mice.ResultsWe found that elderly patients with advanced non-small cell lung cancer (aNSCLC) aged ≥ 75 years exhibited poorer progression-free survival (PFS), overall survival (OS) and a lower clinical response rate after immunotherapy. Mechanistically, we showed that the infiltration of NK cells was significantly reduced in aged mice compared to younger mice. Furthermore, the aged NK cells could also suppress the activation of tumor antigen-specific CD8+ T cells by inhibiting the recruitment and activation of CD103+ dendritic cells (DCs). Adoptive transfer of NK cells from young mice to aged mice promoted TME remodeling, and reversed immunotherapy resistance.ConclusionOur findings revealed the decreased sensitivity of elderly patients to immunotherapy, as well as in aged mice. This may be attributed to the reduction of NK cells in aged mice, which inhibits CD103+ DCs recruitment and its CD86 expression and ultimately leads to immunotherapy resistance.

An ultrasound-activated nanoplatform remodels tumor microenvironment through diverse cell death induction for improved immunotherapy

Although nanomaterial-based nanomedicine provides many powerful tools to treat cancer, most focus on the “immunosilent” apoptosis process. In contrast, ferroptosis and immunogenic cell death, two non-apoptotic forms of programmed cell death (PCD), have been shown to enhance or alter the activity of the immune system. Therefore, there is a need to design and develop nanoplatforms that can induce multiple modes of cell death other than apoptosis to stimulate antitumor immunity and remodel the immunosuppressive tumor microenvironment for cancer therapy. In this study, a new type of multifunctional nanocomposite mainly consisting of HMME, Fe3+ and Tannic acid, denoted HFT NPs, was designed and synthesized to induce multiple modes of cell death and prime the tumor microenvironment (TME). The HFT NPs consolidate two functions into one nano-system: HMME as a sonosensitizer for the generation of reactive oxygen species (ROS) 1O2 upon ultrasound irradiation, and Fe3+ as a GSH scavenger for the induction of ferroptosis and the production of ROS ·OH through inorganic catalytic reactions. The administration of HFT NPs and subsequent ultrasound treatment caused cell death through the consumption of GSH, the generation of ROS, ultimately inducing apoptosis, ferroptosis, and immunogenic cell death (ICD). More importantly, the combination of HFT NPs and ultrasound irradiation could reshape the TME and recruit more T cell infiltration, and its combination with immune checkpoint blockade anti-PD-1 antibody could eradicate tumors with low immunogenicity and a cold TME. This new nano-system integrates sonodynamic and chemodynamic properties to achieve outstanding therapeutic outcomes when combined with immunotherapy. Collectively, this study demonstrates that it is possible to potentiate cancer immunotherapy through the rational and innovative design of relatively simple materials.

Intra-tumoral administration of CHST15 siRNA remodels tumor microenvironment and augments tumor-infiltrating T cells in pancreatic cancer

Intra-tumoral administration of CHST15 siRNA remodels tumor microenvironment and augments tumor-infiltrating T cells in pancreatic cancer

Abstract PO1-06-11: Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer

Abstract Background: Lung metastasis occurs in 30% of triple negative breast cancer (TNBC), the detailed molecular mechanism remains largely unexplored. Accumulating evidence suggests that PD-L1+-tumor associated macrophages (PD-L1+-TAMs) are closely related to immune suppression, invasion, and metastasis. Tumor-derived exosomes have been reported to remodel tumor microenvironment and accelerate metastasis via packing and delivering a variety of biologically active molecules. Our previous studies found that exosomes from breast cancer cells polarized macrophages toward TAMs within the primary tumor nest. The aim of this study was to reveal whether TNBC-derived exosomes were able to polarize lung macrophages towards PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, therefore discovering the underlying mechanism of lung-tropic metastasis. Methods: Exosomes isolated from EO771 TNBC cell line (EO771/exo) and HC11 normal breast epithelial cell line (HC11/exo) were characterized. Uptake of exosomes by macrophages was visualized by confocal microscopy. Mass spectrometry was applied to identify the significantly highly expressed HMGB1 protein in EO771/exo, and ligands of HMGB1 on lung macrophages were detected by co-immunoprecipitation. Functions of EO771/exo and HMGB1 in inducing PD-L1+-TAMs and generating a pre-metastatic immunosuppressive niche were confirmed by both in vitro and in vivo studies. Roles of EO771/exo and HMGB1 on glycolysis and lactate production were evaluated by metabolism assays. Associations between PD-L1+-TAMs and CD3+CD8+ T lymphocytes within TNBC axillary lymph nodes and lung metastasis specimens were analyzed by immunofluorescence and immunohistochemistry. Results: Confocal microscopy showed constant internalization and absorption of EO771/exo by macrophages. EO771/exo could polarize macrophages toward PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, with respect to HC11/exo. Mass spectrometry, clinical samples, and bioinformatics analysis revealed that HMGB1 was highly expressed in EO771/exo and was involved in cellular communication and metabolism processes. Co-immunoprecipitation and 3-dimensional modeling indicated a strong binding of HMGB1 with Tim-3 ligand on lung macrophages. Macrophages treated with EO771/exo displayed an enhanced glycolysis and lactate production. Flow cytometry demonstrated that green fluorescent protein (GFP)-EO771 tumor-bearing mice had more lung micro-metastases than brain and liver. Immunofluorescence and laser confocal microscopy confirmed that EO771/exo, with respect to HC11/exo, were able to increase lung micro-metastases and escalate PD-L1 expression on lung macrophages. Moreover, elevated PD-L1+-TAMs and reduced CD3+CD8+ T lymphocytes were found in TNBC positive axillary lymph nodes and lung metastasis specimens than TNBC negative axillary lymph nodes. Conclusions: Exosomal HMGB1 from TNBC could target Tim-3 ligand on lung macrophages and accelerate glycolysis and lactate production to induce PD-L1+-TAMs, thereby suppressing CD3+CD8+ T lymphocytes immunity and generating a pre-metastasis immune-suppressive niche. Our research would uncover a novel mechanism of lung-tropic metastasis and provide a new therapeutic approach for TNBC treatment. Keywords: breast cancer; lung metastasis; exosomes; tumor-associated macrophages; metabolic reprogramming Citation Format: Wei-Xian Chen, Jia-Hao Wu, Tian-Cheng Cheng, Sujin Yang. Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-11.

IL-7R Expression Correlates with Prognosis in Breast Cancer.

The tumor microenvironment (TME) exerts a significant influence on the development, invasion, metastasis, and drug resistance of breast cancer. Therefore, this study sought to investigate potential prognostic factors and markers indicative of TME remodeling in breast cancer, utilizing data from the TCGA database. In this study, transcriptome RNA-seq data from 1222 breast cancer samples were processed using CIBERSORT and ESTIMATE algorithms. We conducted a differential gene expression analysis utilizing COX regression analysis and constructed protein-protein interaction (PPI) networks for enhanced visualization. Through univariate COX analysis and cross-analysis within PPI networks, the Interleukin-7 receptor (IL-7R) emerged as a potential predictor. Subsequently, we performed a comprehensive investigation encompassing single-gene survival analysis, clinical correlation assessment, and GSEA enrichment analysis targeting IL-7R as a core gene associated with prognosis. We examined the expression of IL-7R in human breast cancer and normal breast tissue through clinical studies and cytology experiments, followed by an indepth analysis of the relationship between IL-7R and breast cancer. The survival analysis revealed that breast cancer patients with elevated IL-7R expression experienced prolonged survival compared to those with lower IL-7R levels. Results obtained from the Wilcoxon rank-sum test, along with clinical and cellular experiments, indicated higher IL-7R expression in tumor samples compared to normal samples. Correlation tests conducted between IL-7R expression and clinicopathological stage characteristics highlighted statistically significant associations between IL-7R expression and the T and M stages. Additionally, cell classification analysis of tumor-infiltrating immune cells (TIC) proportion showed that activated CD4+ T cells and CD8 T cells of memory B cells were positively correlated with IL-7R expression. These findings further underscored the impact of IL-7R levels on the tumor microenvironment (TME). IL-7R emerges as a potential prognostic indicator for breast cancer patients, particularly in sustaining the immunoactive status of the tumor microenvironment (TME) and contributing to immune reconstitution. These findings offer novel insights into breast cancer treatment strategies.

Targeting metabolism of breast cancer and its implications in T cell immunotherapy.

Breast cancer is a prominent health issue amongst women around the world. Immunotherapies including tumor targeted antibodies, adoptive T cell therapy, vaccines, and immune checkpoint blockers have rejuvenated the clinical management of breast cancer, but the prognosis of patients remains dismal. Metabolic reprogramming and immune escape are two important mechanisms supporting the progression of breast cancer. The deprivation uptake of nutrients (such as glucose, amino acid, and lipid) by breast cancer cells has a significant impact on tumor growth and microenvironment remodeling. In recent years, in-depth researches on the mechanism of metabolic reprogramming and immune escape have been extensively conducted, and targeting metabolic reprogramming has been proposed as a new therapeutic strategy for breast cancer. This article reviews the abnormal metabolism of breast cancer cells and its impact on the anti-tumor activity of T cells, and further explores the possibility of targeting metabolism as a therapeutic strategy for breast cancer.

Remodeling of Tumor Microenvironment Induced by Chemoradiation in Esophageal Cancer

Remodeling of Tumor Microenvironment Induced by Chemoradiation in Esophageal Cancer

Abstract LB190: Cancer-stromal cells interaction dominates the expression of pro-tumor decreted protein chitinase-3-like-1 in pancreatic cancer

Abstract In the context of the tumor microenvironment (TME), a substantial increased secreted protein, chitinase-3 like-protein-1 (CHI3L1) has been observed, emanating from various cell types such as cancer cells, macrophages, and T cells. The development of an immunosuppressive and fibrotic TME has been implicated in CHI3L1-mediated cancer progression. Despite recognizing the potential for CHI3L1 blockade to facilitate cancer regression, there remains a limited understanding of the regulatory mechanisms governing the expression of this tumor-promoting protein within cancer cells. Our investigations, along with others, have identified low levels of CHI3L1 protein in normal epithelial cells, in stark contrast to its abundant presence in pancreatic cancer tumor specimens. We propose that the interaction between cancer and stromal cells in the TME induces CHI3L1 expression within cancer cells. To test this hypothesis, we established a co-culture system involving cancer and stromal cells including pancreatic stellate cells, macrophages and T cells to emulate the TME of pancreatic cancer. Subsequently, we observed a ten-fold increase in CHI3L1 expression in cancer cells upon exposure to co-culture media. Through secretomic analysis, C-X-C motif chemokine ligand 10 (CXCL10) emerged as a potential inducer of CHI3L1 expression in response to co-culture media. CXCL10/CXCR3 signaling has been reported to correlate with poor prognosis in pancreatic cancer, while their role in disease development is still unclear. Concurrently, Gene Set Enrichment Analysis (GSEA) identified the activation of TNFα signaling via NF-κB and TGF-β pathways in pancreatic cancer patients with high CHI3L1 expression. Notably, chromatin-immunoprecipitation and promoter activity assays validated the direct binding and transcriptional regulatory role of NF-κB, a downstream transcription factor of CXCL10, on CHI3L1 gene in cancer cells. In addition, epigenetic reprogramming occurred during co-culture media education. Our observations indicated demethylation of the CHI3L1 promoter during co-culture media exposure, a phenomenon confirmed to be mediated by STAT3 activation. In summary, this study elucidates, for the first time, the intricate crosstalk between cancer and stromal cells within the TME of pancreatic cancer as a pivotal factor for the transcriptional and epigenetic dysregulation of CHI3L1, which then contributes to TME remodeling and malignant pancreatic cancer progression. Citation Format: Pei-Chia (Peggy) Su, Yi-Ching Wang. Cancer-stromal cells interaction dominates the expression of pro-tumor decreted protein chitinase-3-like-1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB190.

Abstract LB319: Klf5 acetylation remodels tumor microenvironment to constrain PTEN-deficient prostate tumor growth

Abstract Prostate cancer is the most common cancer and the second leading cause of cancer-related death in men in the United States. PTEN deficiency is prevalent in patients with prostate cancer. As a tumor suppressor, PTEN is mutated in approximately 20% of primary prostate cancers, and up to 50% of metastatic castration resistant prostate cancer. PTEN inactivation results in high-grade adenocarcinoma with a long latency. The limited tumor progression induced by PTEN deficiency suggests that additional molecular events are activated to constrain tumor progression, and second hits are required. Understanding these secondary genetic hits will provide the rationale for combined therapies in prostate cancer treatment. In this study, we employed a unique genetically knockin mouse model and found that PTEN deficiency induces a KLF5 K369 acetylation-dependent barrier that attenuates FGFR1 signaling and constrains prostate tumor growth. More importantly, we discovered novel microenvironmental crosstalk between prostate cancer cells and cancer associated fibroblasts (CAFs). As suggested by RNA-seq and bioinformatic analyses and further validated by molecular and cellular experiments, we found that interruption of KLF5 acetylation in cancer cells stimulates CAFs to release FGF9 via TNF-α, and FGF9 activating FGFR1 signaling in cancer cells. In addition to this paracrine crosstalk, deacetylated KLF5 induces CX3CR1 and facilitates FGFR1 activation. Our findings provide a proof of concept for posttranslational modifications as essential molecular events induced by PTEN inactivation to stall prostate cancer progression and demonstrate KLF5 acetylation as a key component in a negative feedback loop by which FGFR1 was suppressed by AKT activation. This study reveals a paracrine reciprocal communication between cancer cells and fibroblasts and provides a clinical rationale for combined therapies using FGFR1 inhibitors (or CX3CR1 inhibitors) and p-AKT inhibitors in PTEN-deficient prostate cancer. Citation Format: Baotong Zhang, Mingcheng Liu, Fengyi Mai, Siyuan Xia, Jin-Tang Dong. Klf5 acetylation remodels tumor microenvironment to constrain PTEN-deficient prostate tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB319.

Nanomedicine Remodels Tumor Microenvironment for Solid Tumor Immunotherapy.

Although immunotherapy is relatively effective in treating hematological malignancies, their efficacy against solid tumors is still suboptimal or even noneffective presently. Compared to hematological cancers, solid tumors exhibit strikingly different immunosuppressive microenvironment, severely deteriorating the efficacy of immunotherapy: (1) chemical features such as hypoxia and mild acidity suppress the activity of immune cells, (2) the pro-tumorigenic domestication of immune cells in the microenvironment within the solid tumors further undermines the effectiveness of immunotherapy, and (3) the dense physical barrier of solid tumor tissues prevents the effective intratumoral infiltration and contact killing of active immune cells. Therefore, we believe that reversing the immunosuppressive microenvironment are of critical priority for the immunotherapy against solid tumors. Due to their unique morphologies, structures, and compositions, nanomedicines have become powerful tools for achieving this goal. In this Perspective, we will first briefly introduce the immunosuppressive microenvironment of solid tumors and then summarize the most recent progresses in nanomedicine-based immunotherapy for solid tumors by remodeling tumor immune-microenvironment in a comprehensive manner. It is highly expected that this Perspective will aid in advancing immunotherapy against solid tumors, and we are highly optimistic on the future development in this burgeoning field.

Combination Nano-Delivery Systems Remodel the Immunosuppressive Tumor Microenvironment for Metastatic Triple-Negative Breast Cancer Therapy.

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer for which effective therapies are lacking. Targeted remodeling of the immunosuppressive tumor microenvironment (TME) and activation of the body's immune system to fight tumors with well-designed nanoparticles have emerged as pivotal breakthroughs in tumor treatment. To simultaneously remodel the immunosuppressive TME and trigger immune responses, we designed two potential therapeutic nanodelivery systems to inhibit TNBC. First, the bromodomain-containing protein 4 (BRD4) inhibitor JQ1 and the cyclooxygenase-2 (COX-2) inhibitor celecoxib (CXB) were coloaded into chondroitin sulfate (CS) to obtain CS@JQ1/CXB nanoparticles (NPs). Then, the biomimetic nanosystem MM@P3 was prepared by coating branched polymer poly(β-amino ester) self-assembled NPs with melittin embedded macrophage membranes (MM). Both in vitro and in vivo, the CS@JQ1/CXB and MM@P3 NPs showed excellent immune activation efficiencies. Combination treatment exhibited synergistic cytotoxicity, antimigration ability, and apoptosis-inducing and immune activation effects on TNBC cells and effectively suppressed tumor growth and metastasis in TNBC tumor-bearing mice by activating the tumor immune response and inhibiting angiogenesis. In summary, this study offers a novel combinatorial immunotherapeutic strategy for the clinical TNBC treatment.

Abstract 6957: Extrachromosomal FGFR2 is delivered in extracellular vesicles in two models of cancer of unknown primary

Abstract The extracellular vesicle (EV) route is essential for cell-to-cell communication. Cancer cells release EVs in the extracellular space, where they can interact with recipient cells inducing regulation of gene expression, activation of specific signaling pathways and tumor microenvironment remodeling. We investigated the EVs released by two cell lines of cancer of unknown primary site (CUP) that we established from patient’s tumor cells, namely CUP#55 and CUP#96, as described in https://www.medrxiv.org/content/10.1101/2023.03.12.23287041v1). CUP comprises 3-5% of new cancer cases and presents with metastases of unknown or uncertain origin and no apparent primary tumor. Our CUP cell lines are both characterized by FGFR2 gene amplification. It has been recognized that tumors use circular extrachromosomal DNA (ecDNA) as a way to increase oncogenic amplification. We hypothesized that FGFR2 amplification in CUP cell lines could be associated with ecDNA generation and that this ecDNA could be loaded as cargo inside EVs. FGFR2 copy number was quantified using a probe-based droplet digital PCR assay then we identified the different nature of FGFR2 amplification in the two models using FGFR2 FISH assay: CUP#55 presented a chromosomal homogeneously staining region while CUP#96 displayed double minute chromosomes. FGFR2 DNA was detectable in the cytoplasm fraction, therefore we isolated extracellular vesicles from culture medium using an ultracentrifugation-based protocol and confirmed the presence of the entire length of FGFR2 gene inside small and large vesicles. Since ecDNA contributes to cancer genome remodeling also forming chimeric circles, we confirmed the circular nature of a fraction of FGFR2 ecDNA using the Plasmid-Safe ATP-dependent DNAse test. Finally, we tested the oncogene delivery capability of CUP EVs by administering CUP#96 and CUP#55 small and large vesicles to recipient cell lines. We observed an increase of FGFR2 DNA at 24 hours upon EV treatments, and its functional transcription in FGFR2 mRNA after 48 hours. In conclusion, we identified a model of oncogene amplification and delivery in two cell lines of cancer of unknown primary, which could explain the high metastatic potential of this cancer type. (The research leading to these results has received funding from AIRC under IG 2021 - ID. 25789 project - P.I. Ferracin Manuela) Citation Format: Irene Salamon, Gianluca Storci, Beatrice Fontana, Salvatore Serravalle, Giulia Gallerani, Roberta Roncarati, Spartaco Santi, Massimiliano Bonafè, Manuela Ferracin. Extrachromosomal FGFR2 is delivered in extracellular vesicles in two models of cancer of unknown primary [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 6957.

Abstract 3612: Preclinical & clinical activity of autologous mRNA engineered chimeric antigen receptor monocytes for targeted cancer immunotherapy

Abstract Introduction: The tumor microenvironment (TME) is a dynamic and multifaceted system that comprises largely myeloid lineage immune cells including neutrophils, macrophages, dendritic cells and monocytes. When activated, these cells are can effectively infiltrate TME and orchestrate anti-tumor immune response. Reprogramming myeloid cells by arming them with a tumor specific chimeric antigen receptor (CAR) presents an attractive strategy to overcome current limitations in solid tumor treatment. In this study, we present the isolation and mRNA-based engineering of monocytes with chimeric antigen receptors (CARs), followed by their in vivo application for cancer treatment in rodent models and in human T cell lymphoma patients. Results: Engineered monocytes demonstrated the abilities to infiltrate the TME, undergo activation, and differentiate into macrophages and dendritic cells. Upon CAR activation, these reprogrammed myeloid cells utilized both direct and indirect mechanisms to eliminate tumor cells. This included direct tumor cell killing through phagocytosis and TNF-associated mechanisms. Furthermore, engineered monocytes exhibited the capacity to recruit natural killer (NK) cells and T cells to the TME. In the case of T cells, these engineered myeloid cells facilitated the presentation of neoantigens to T cells and stimulate an adaptive immune response. In patients with T cell lymphoma, treatment with engineered monocytes expressing a CAR targeting CD5 exhibited a favorable safety profile and was associated with TME remodeling, expansion of novel T cell clones, and promising survival outcomes. Conclusions: These findings underscore the potential of reprogrammed myeloid cells as a novel avenue for cancer immunotherapy, offering hope for improved treatments and outcomes in the fight against cancer. Citation Format: Yuxiao Wang, Michele Gerber, Michael Gorgievski, Josephine D'Alessandro, Neha Diwanji, Ronald Vale, Siddhartha Mukherjee, Daniel Getts. Preclinical & clinical activity of autologous mRNA engineered chimeric antigen receptor monocytes for targeted cancer immunotherapy [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 3612.

Abstract 2534: Single-cell dissection of heterogeneity and dynamics of tumor ecosystem upon immune-checkpoint blockade in hepatocellular carcinoma

Abstract Background: Therapy-induced alteration of tumor ecosystem poses a major barrier for cancer regression. As the majority of patients with hepatocellular carcinoma (HCC) develops resistance to immune checkpoint blockade (ICB) therapy, we aimed to characterize heterogeneity and dynamics of multicellular tumor ecosystem following anti-PD-1 treatment using scRNA-seq. Methods: In a phase II clinical trial of pembrolizumab on advanced hepatitis B virus-related HCC patients (NCT03419481), we performed a comprehensive single-cell transcriptomic profiling of biopsy samples from 26 patients before and during treatment. The composition and distribution of immune aggregates in responsive tumors was characterized by scRNA-seq and spatial transcriptome analyses. The anti-tumor efficacy of combined therapy with anti-PD-1 and inhibition of immunosuppressive macrophage were further determined in RIL-175-derived PD-1-resistant mouse model. Results: After initial data processing and quality control, we obtained 353,329 high-quality cells that included tumor cells, myeloid cells, lymphoid cells, endothelial cells and fibroblasts. Our analysis revealed that baseline tumor-reactive CD8+T cells exhibited a strong association with improved prognosis. Notably, treatment-induced expansion and differentiation of tumor-reactive CD8+T cells with memory-like phenotype highly correlated with durable ICB response. Moreover, we observed a co-occurrence pattern of key anti-tumor immune subsets in the tumor microenvironment (TME), which associated with ligand-receptor pair interactions. Spatial transcriptome analysis further validated the presence of immune aggregates in responsive tumors. Importantly, we derived an responsive signature from immune aggregates, which showed a strong association with improved prognosis in pan-cancer ICB datasets. Paired comparisons of pre- and on-treatment macrophage percentages in non-responders revealed a specific expansion of an immunosuppressive macrophage subset. Inhibition of this macrophage subset could overcome ICB resistance by enhancing T cell function in our PD-1 resistant model. We further observed that a tumorous epithelial-mesenchymal transition (EMT) program might contribute to therapeutic resistance via TME remodeling. Conclusions: Our study suggests that tumor-reactive CD8+T cells and immune aggregates may be associated with durable ICB response, while ICB-induced upregulation of immunosuppressive macrophages and EMThi tumor cells contributed to therapeutic evasion. These findings provide valuable insights for identification of predictive biomarkers and therapeutic strategies for ICB therapy of HCC. This project is supported by the General Research Fund (14119023, 14120621 and 14115820), the Li Ka Shing Foundation, and the CUHK Strategic Seed Funding for Collaborative Research Scheme. Citation Format: Alfred S. L. Cheng, Zhewen Xiong, Haoran Wu, Jianquan Cao, Zhixian Liang, Jingying Zhou, Joseph Jao-Yiu Sung, Kevin Yip, Stephen Chan. Single-cell dissection of heterogeneity and dynamics of tumor ecosystem upon immune-checkpoint blockade in hepatocellular carcinoma [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 2534.

Abstract 3187: Enhancing STING activation by cyclic dinucleotide-manganese particles for systemic cancer immunotherapy

Abstract Background: Activating the innate immune pathway of stimulator of interferon genes (STING) can elicit potent anti-tumor immunity via the production of type-I interferons (IFN-I). However, cyclic-dinucleotide (CDN) STING agonists have undesirable pharmacological properties. Most CDNs in clinical development are administered intratumorally, thus precluding their utility against advanced cancer. Here, we report the development of a new nanoparticle system for the systemic delivery of CDN STING agonists with potent efficacy, favorable pharmaceutical properties, and acceptable safety profiles. Methods: Various metal ions were screened for synergy with CDNs for IFN-I response from bone marrow-derived dendritic cells. Lipid nanoparticles carrying CDN and manganese ions (termed SNP) were synthesized. Mice bearing syngenetic tumors (including CT26 and B16F10) as well as genetically engineered mouse models were treated by intravenous administration of SNP, followed by tumor monitoring and immune profiling of the tumor microenvironment. White New Zealand rabbits bearing VX2 tumors as well as healthy mongrel dogs were treated with SNP and evaluated. Lastly, human tumor biopsies derived from head and neck squamous cell carcinoma (HNSCC) patients were incubated with SNP or soluble STING agonists and analyzed for immune activation. Results: We identified that Mn2+ achieved strong synergy with CDN STING agonists for inducing IFN-I production. SNP carrying CDN and Mn2+ was dosed intravenously in mice, leading to robust IFN-I response, remodeling of the immunosuppressive tumor microenvironment, and expansion of anti-tumor CD8+ T cells. Intravenous SNP therapy resulted in robust anti-tumor efficacy in multiple murine tumor models, including CT26, B16F10, and a genetically engineered mouse model of MMTV-PyMT triple-negative breast cancer. We have also observed robust anti-tumor efficacy of SNP against VX2 squamous carcinomas in rabbits and have demonstrated the safety of intravenous SNP therapy in healthy dogs. Mechanistically, the efficacy of SNP therapy was dependent on the host STING expression but independent of the tumor STING expression. In addition, the anti-tumor efficacy of SNP was decreased in mice lacking Ifnar1 or Ifngr1 expression in the B16F10 melanoma model, showing the crucial effects of IFN-I and IFN-II responses. Moreover, SNP treatment led to robust IFN-I responses from fresh human tumor biopsies derived from HNSCC patients. Conclusions: SNP empowers highly effective systemic cancer immunotherapy via nanotechnology. It also underscores the potential of utilizing metal ions for immune-mediated disease treatment. Citation Format: Xingwu Zhou, Xiaoqi Sun, Wang Gong, Ziye Wan, Teresa Krieger-Burke, Vilma Yuzbasiyan-Gurkan, Yu Leo Lei, James Moon. Enhancing STING activation by cyclic dinucleotide-manganese particles for systemic cancer immunotherapy [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 3187.

Abstract 3937: Investigating the role of fibulin 3 in pancreatic tumorigenesis

Abstract Pancreatic ductal adenocarcinoma (PDA) is a highly lethal malignancy with a five-year survival rate of less than 11%, making it one of the deadliest cancers. Notably, 95% of PDA cases exhibit KRAS mutations, contributing to highly dysregulated cell signaling networks. Despite extensive research into the interplay of various oncogenic pathways in pancreatic tumorigenesis, targeted therapies against these signaling networks have displayed modest efficacy, with most patients rapidly developing therapeutic resistance. Recent studies highlight the significance of the carbohydrate antigen 19-9 (CA19-9) as a functional biomarker linked to disease progression and resistance. However, understanding the detailed functions of CA19-9-modified proteins in PDA has been limited due to the lack of adequate models for synthesizing CA19-9. To address this challenge, we have developed unique mouse and 3D organoid culture models capable of producing CA19-9. CA19-9 elevation in mice with KRAS-mutations results in an aggressive PDA phenotype through increased tumor proliferation and microenvironment (TME) remodeling. Furthermore, our research has identified Fibulin 3 (FBLN3), an extracellular matrix glycoprotein, as a secreted, CA19-9-modified protein that stimulates tumorigenesis. In this study, we found that FBLN3 not only facilitates tumor proliferation but also drives epithelial-mesenchymal transition (EMT) and promotes STAT3 pathways activation in the CA19-9pos, KRAS-mutant PDA organoids. Additionally, in both CA19-9pos, KRAS-mutant PDA mouse models and human PDA, FBLN3 is expressed by cancer-associated fibroblasts (CAFs), suggesting its role in TME remodeling. These findings have significant implications for providing novel insights into PDA biology and paracrine signaling mechanisms, identifying FBLN3 as a potential therapeutic target for the improved treatment of this devastating disease. Citation Format: Hyemin Song, Jasper Hsu, Xiaoxue Lin, Satoshi Ogawa, Vasiliki Pantazopoulou, Kristina Peck, Chelsea Bottomley, Shira Okhovat, McKenna Stamp, Kassidy Curtis, Jeffrey D. Esko, Dannielle D. Engle. Investigating the role of fibulin 3 in pancreatic tumorigenesis [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 3937.