Tumor-promoting Microenvironment Research Articles

Abstract B075: Functional characterization of TGF-β-dependent cancer-associated fibroblasts in pancreatic cancer using novel in vitro and in vivo models

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant cancer- associated fibroblasts (CAFs) which contribute to a tumor-promoting, chemotherapy-resistant and immunosuppressive tumor microenvironment (TME). However, CAF-ablating therapeutic strategies have thus far failed in clinical trials. One potential reason is that PDAC CAFs are heterogeneous and plastic. It was recently discovered that myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs) can originate from pancreatic stellate cells (PSCs) via TGF-β and IL-1 signaling, respectively. However, their functional heterogeneity remains to be elucidated. We hypothesize that distinct CAF subtypes exhibit different tumor-promoting roles mediated by targetable signaling mechanisms. To this end, we aimed at characterizing the functions of TGF- β-dependent myCAFs in shaping the TME and influencing PDAC progression. Methods: We generated novel myCAF-depleted PDAC mouse models by orthotopically injecting PDAC organoids derived from KPC (Kras LSL-G12D/+ ; Trp53 LSL-R172H/+ ; Pdx1-Cre) mice into Fap-Tgfbr2 knockout (Fap-tTA; Rosa26 LSLtdTom/+ ; tet-O-Cre; Tgfbr2 fl/fl ) mice. The pancreatic tumors were analyzed by flow cytometry, histology and single-cell RNA-sequencing (scRNAseq). In a complementary approach, we generated myCAF-enriched PDAC mouse models by orthotopically co-injecting KPC PDAC organoids and novel TGF-β-driven ‘myCAF-locked’ murine PSCs. In addition, to further dissect myCAF-malignant cell signaling crosstalk, KPC PDAC organoids and myCAF-locked PSCs were co-cultured and then flow-sorted for bulk RNA- sequencing. Results: MyCAF-depleted PDAC tumors from Fap-Tgfbr2 knockout mice had a significant decrease in myCAFs, and a significant expansion of iCAFs, compared to control mice. Additionally, Masson’s trichrome staining indicated significant reduction in collagen content in the TME upon myCAF depletion. Moreover, scRNAseq analysis revealed that myCAF depletion shaped the composition of innate immune cells, including macrophages and neutrophils. Notably, although primary tumor growth was not altered, liver and lung metastases decreased, while diaphragm metastases and ascites increased, in myCAF-depleted PDAC mice. Conversely, liver and lung metastases increased, while diaphragm metastases and ascites decreased in myCAF-enriched PDAC mouse models, suggesting that TGF-β-dependent myCAFs play a role in promoting distant metastases. In line with this, transcriptomic analysis of PDAC organoids co-cultured with myCAF-locked PSCs showed enrichment of pathways in epithelial-mesenchymal transition, hypoxia, VEGF, WNT and cell-matrix interactions. Conclusion: Our study demonstrates that TGF-β-dependent CAFs are not only largely responsible for collagen deposition in PDAC, but also possess immune-modulating properties. Moreover, our study supports a role of TGF-β-dependent CAFs in promoting PDAC metastasis to the liver and the lungs, potentially through upregulating pro-metastatic pathways in the malignant cells. Citation Format: Priscilla S.W. Cheng, Muntadher Jihad, Judhell S. Manansala, Weike Luo, Gianluca Mucciolo, Sara Pinto Teles, Giulia Biffi. Functional characterization of TGF-β-dependent cancer-associated fibroblasts in pancreatic cancer using novel in vitro and in vivo models [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 B075.

B-cell intrinsic RANK signaling cooperates with TCL1 to induce lineage-dependent B-cell transformation

B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), remain incurable, with MM particularly prone to relapse. Our study introduces a novel mouse model with active RANK signaling and the TCL1 oncogene, displaying both CLL and MM phenotypes. In younger mice, TCL1 and RANK expression expands CLL-like B1-lymphocytes, while MM originates from B2-cells, becoming predominant in later stages and leading to severe disease progression and mortality. The induced MM mimics human disease, exhibiting features like clonal plasma cell expansion, paraproteinemia, anemia, and kidney and bone failure, as well as critical immunosurveillance strategies that promote a tumor-supportive microenvironment. This research elucidates the differential impacts of RANK activation in B1- and B2-cells and underscores the distinct roles of single versus combined oncogenes in B-cell malignancies. We also demonstrate that human MM cells express RANK and that inhibiting RANK signaling can reduce MM progression in a xenotransplantation model. Our study provides a rationale for further investigating the effects of RANK signaling in B-cell transformation and the shaping of a tumor-promoting microenvironment.

Enhanced RHO-ROCK signaling is associated with CRELD2 production and fibroblast recruitment in cutaneous squamous cell carcinoma.

A key characteristic of cancer cells is their ability to induce changes in their microenvironment that render it permissive to tumor growth, invasion and metastasis. Indeed, these changes are required for tumor progression. Consequently, the tumor microenvironment is emerging as a key source of new targets against cancer, with novel therapies aimed at reversing tumor-promoting changes, reinstating a tumor-hostile microenvironment and suppressing disease progression. RHO-ROCK signaling, and consequent tension within the cellular actomyosin cytoskeleton, regulates a paracrine signaling cascade that establishes a tumor-promoting microenvironment. Here, we show that consistent with our observations in breast cancer, enhanced ROCK activity and consequent production of CRELD2 is associated with the recruitment and tumor-promoting polarization of cancer-associated fibroblasts in cutaneous squamous cell carcinoma. Our observations provide support for the notion that the role of RHO-ROCK signaling in establishing a tumor-promoting microenvironment may be conserved across patients and potentially also different cancer types.

Single-cell transcriptome dissecting the microenvironment remodeled by PD1 blockade combined with photodynamic therapy in a mouse model of oral carcinogenesis.

Oral squamous cell carcinoma (OSCC) stands as a predominant and perilous malignant neoplasm globally, with the majority of cases originating from oral potential malignant disorders (OPMDs). Despite this, effective strategies to impede the progression of OPMDs to OSCC remain elusive. In this study, we established mouse models of oral carcinogenesis via 4-nitroquinoline 1-oxide induction, mirroring the sequential transformation from normal oral mucosa to OPMDs, culminating in OSCC development. By intervening during the OPMDs stage, we observed that combining PD1 blockade with photodynamic therapy (PDT) significantly mitigated oral carcinogenesis progression. Single-cell transcriptomic sequencing unveiled microenvironmental dysregulation occurring predominantly from OPMDs to OSCC stages, fostering a tumor-promoting milieu characterized by increased Treg proportion, heightened S100A8 expression, and decreased Fib_Igfbp5 (a specific fibroblast subtype) proportion, among others. Notably, intervening with PD1 blockade and PDT during the OPMDs stage hindered the formation of the tumor-promoting microenvironment, resulting in decreased Treg proportion, reduced S100A8 expression, and increased Fib_Igfbp5 proportion. Moreover, combination therapy elicited a more robust treatment-associated immune response compared with monotherapy. In essence, our findings present a novel strategy for curtailing the progression of oral carcinogenesis.

DIPG-86. INVESTIGATING THE TUMOR ENVIRONMENT AND SIGNALING DEPENDENCIES DRIVING DIFFUSE MIDLINE GLIOMA TUMOR INITIATION, PROGRESSION, AND RESISTANCE: A COMPREHENSIVE APPROACH LEVERAGING BULK MULTIOMICS DECONVOLUTION AND SINGLE-CELL OMICS VALIDATION

Abstract BACKGROUND Diffuse Midline Glioma (DMG) is a incurable tumor affecting children. Recent genomic investigations have identified a recurrent H3K27M mutation which induces global alterations in histone methylation patterns and DNA methylation. These epigenetic modifications hint at pivotal roles in DMG pathogenesis, yet effective therapeutic strategies remain elusive, with median survival rates stagnant at approximately one year. This shortfall stems from two main factors: 1) Inadequate multiomics studies hindering our understanding of DMG evolution and tumor progression, and 2) Limited comprehension of the tumor microenvironment in DMG. METHODS To elucidate DMG’s clonal evolution, we adopted a comprehensive multi-region sampling approach, acquiring 33 specimens from seven patients. Whole-exome and transcriptome sequencing, alongside DNA methylation profiling, were performed. Additionally, we generated one of the most extensive single-cell multiomics datasets (>300,000 cells from 21 additional samples), with a focus on pre- and post-treatment, and dissemination effects in DMG. RESULTS Our analysis delineated a tumor-promoting microenvironment characterized by hypoxia and pro-inflammatory conditions, nurturing genomic alterations and specialized biological processes such as proliferation and epithelial-mesenchymal transition. Notably, a majority of immune cells exhibited M1-like polarization, bolstering pro-inflammatory programs within the tumor milieu. Subclones within the DMG cohort demonstrated dissemination potential, often exhibiting up-regulation of NOTCH, P53, and WNT beta-catenin signaling pathways. These findings suggest that DMG clones harboring dissemination capabilities outside the pons acquire additional phenotypic features, possibly mediated by epigenetic or transcriptional alterations, contributing to enhanced migratory and aggressive behaviors. CONCLUSIONS Our study unveils the parallel evolution of DMG at genetic, epigenetic, and transcriptional levels, unveiling novel subclonal phenotypes governing tumor behavior. Crucially, we identify critical environmental shifts such as hypoxia and inflammatory changes, coupled with specialized signaling programs, driving dissemination and resistance phenotypes. These insights may pave the way for generating accurate genetically mouse models and targeted therapeutic interventions to combat this challenging malignancy.

Systemic versus localized Bacillus Calmette Guérin immunotherapy of bladder cancer promotes an anti-tumoral microenvironment: Novel role of trained immunity.

Treatment for higher-risk non-muscle invasive bladder cancer (NMIBC) involves intravesical immunotherapy with Bacillus Calmette Guérin (BCG); however, disease recurrence and progression occur frequently. Systemic immunity is critical for successful cancer immunotherapy; thus, recurrence of NMIBC may be due to suboptimal systemic activation of anti-tumor immunity after local immunotherapy. We previously reported that systemically acquired trained immunity (a form of innate immune memory) in circulating monocytes is associated with increased time-to-recurrence in patients with NMIBC treated with BCG. Herein, we used a mouse model of NMIBC to compare the effects of intravesical versus intravenous (systemic) BCG immunotherapy on the local and peripheral immune microenvironments. We also assessed whether BCG-induced trained immunity modulates anti-tumor immune responses. Compared with intravesical BCG, which led to a tumor-promoting immune microenvironment, intravenous BCG resulted in an anti-tumoral bladder microenvironment characterized by increased proportions of cytotoxic T lymphocytes (CTLs), and decreased proportions of myeloid-derived suppressor cells. Polarization toward anti-tumoral immunity occurred in draining lymph nodes, spleen, and bone marrow following intravenous versus intravesical BCG treatment. Pre-treatment with intravesical BCG was associated with increased rate of tumor growth compared with intravenous BCG pre-treatment. Trained immunity contributed to remodeling of the tumor immune microenvironment, as co-instillation of BCG-trained macrophages with ovalbumin-expressing bladder tumor cells increased the proportion of tumor-specific CTLs. Furthermore, BCG-trained dendritic cells exhibited enhanced antigen uptake and presentation and promoted CTL proliferation. Our data support the concept that systemic immune activation promotes anti-tumor responses, and that BCG-induced trained immunity is important in driving anti-tumor adaptive immunity.

Deficiency of factor-inhibiting HIF creates a tumor-promoting immune microenvironment

Hypoxia signaling influences tumor development through both cell-intrinsic and -extrinsic pathways. Inhibiting hypoxia-inducible factor (HIF) function has recently been approved as a cancer treatment strategy. Hence, it is important to understand how regulators of HIF may affect tumor growth under physiological conditions. Here we report that in aging mice factor-inhibiting HIF (FIH), one of the most studied negative regulators of HIF, is a haploinsufficient suppressor of spontaneous B cell lymphomas, particular pulmonary B cell lymphomas. FIH deficiency alters immune composition in aged mice and creates a tumor-supportive immune environment demonstrated in syngeneic mouse tumor models. Mechanistically, FIH-defective myeloid cells acquire tumor-supportive properties in response to signals secreted by cancer cells or produced in the tumor microenvironment with enhanced arginase expression and cytokine-directed migration. Together, these data demonstrate that under physiological conditions, FIH plays a key role in maintaining immune homeostasis and can suppress tumorigenesis through a cell-extrinsic pathway.

Extracellular vesicles released by cancer-associated fibroblast-induced myeloid-derived suppressor cells inhibit T-cell function

ABSTRACT Myeloid cells are known to play a crucial role in creating a tumor-promoting and immune suppressive microenvironment. Our previous study demonstrated that primary human monocytes can be polarized into immunosuppressive myeloid-derived suppressor cells (MDSCs) by cancer-associated fibroblasts (CAFs) in a 3D co-culture system. However, the molecular mechanisms underlying the immunosuppressive function of MDSCs, especially CAF-induced MDSCs, remain poorly understood. Using mass spectrometry-based proteomics, we compared cell surface protein changes among monocytes, in vitro differentiated CAF-induced MDSCs, M1/M2 macrophages, and dendritic cells, and identified an extracellular vesicle (EV)-mediated secretory phenotype of MDSCs. Functional assays using an MDSC/T-cell co-culture system revealed that blocking EV generation in CAF-induced MDSCs reversed their ability to suppress T-cell proliferation, while EVs isolated from CAF-induced MDSCs directly inhibited T-cell function. Furthermore, we identified fructose bisphosphatase 1 (FBP1) as a cargo protein that is highly enriched in EVs isolated from CAF-induced MDSCs, and pharmacological inhibition of FBP1 partially reversed the suppressive phenotype of MDSCs. Our findings provide valuable insights into the cell surface proteome of different monocyte-derived myeloid subsets and uncover a novel mechanism underlying the interplay between CAFs and myeloid cells in shaping a tumor-permissive microenvironment.

The ways Fusobacterium nucleatum translocate to breast tissue and contribute to breast cancer development.

Breast cancer is among the most prevalent malignancies in women worldwide. Epidemiological findings suggested that periodontal diseases may be associated with breast cancer, among which Fusobacterium nucleatum is considered an important cross-participant. In this work, we comprehensively summarize the known mechanisms of how F. nucleatum translocates to, colonizes in mammary tumors, and promotes the carcinogenesis. Specifically, F. nucleatum translocates to mammary tissue through the mammary-intestinal axis, direct nipple contact, and hematogenous transmission. Subsequently, F. nucleatum takes advantage of fusobacterium autotransporter protein 2 to colonize breast cancer and uses virulence factors fusobacterium adhesin A and lipopolysaccharide to promote proliferation. Moreover, the upregulated matrix metalloproteinase-9 induced by F. nucleatum does not only trigger the inflammatory response but also facilitates the tumor-promoting microenvironment. Aside from the pro-inflammatory effect, F. nucleatum may also be engaged in tumor immune evasion, which is achieved through the action of virulence factors on immune checkpoint receptors highly expressed on T cells, natural killer cells, and tumor-infiltrating lymphocytes. Taking breast cancer as an example, more relevant research studies may expand our current knowledge of how oral microbes affect systemic health. Hopefully, exploring these mechanisms in depth could provide new strategies for safer and more effective biologic and targeted therapies targeted at breast cancer.

High Level of IL-16 Secreted By Non-GCB DLBCL Facilitates Tumor Growth Though the Tumor-Promoting and Immunosuppressive Tumor Microenvironment

Introduction: Diffuse Large B-Cell Lymphoma (DLBCL) is a highly heterogeneous non-Hodgkin's lymphoma. Based on cell origin, DLBCL is classified into two main molecular subtypes: the germinal center B-cell-like (GCB) subtype and the activated B-cell-like (ABC) subtype. ABC subtype is more aggressive and associated with a worse prognosis than GCB subtype. Previous studies have shown that cytokines play an important role in the growth of ABC subtypes, and IL-16 was supposed to be highly expressed in ABC-DLBCL, however it has not been explored in depth in DLBCL. In this study, we broadly assessed the expression and role of IL-16 on DLBCL. Method: Serum and tissues samples from patients with DLBCL and whole blood from healthy donors were collected. The characterization of protein expression, mRNA expression, and levels of cytokines in cells and tissues was detected by Western blotting, IHC/IHF, PCR, ELISA, RNA-seq, and Flow cytometry. Cells experiments such as cell proliferation, cell apoptosis, cell cycle, cell migration, and ADPC were detected by CCK-8, Flow cytometry, and Transwells. Gene knockdown or overexpression was conducted by lentiviruses and then verified by Western blotting and ELISA. We established subcutaneous tumors (human DLBCL cells and murine DLBCL cells) in BALB/c mice, BALC/c nude mice, and NOD SCID mice for the tumor size or the therapeutic evaluation. Results: First, we found that IL-16, secreted by the cleavage of active caspase-3 from the lymphoma cells under tumor burden, was highly expressed in ABC-DLBCL compared to GCB-DLBCL. The serum IL-16 levels were positively correlated with unfavorable prognosis in DLBCL patients. The dynamic change of serum IL-16 levels accurately predicted the treatment response in patients with DLBCL who received R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone) or anti-CD19 Chimeric Antigen Receptor (CAR) T cells. Interestingly, bioactive IL-16 showed rare effect on DLBCL cells in vitro, but significantly promoted their growth in vivo. Mechanically, tumor-derived bioactive IL-16 promoted the migration of monocytes via CD4+ receptor, thereby inducing the enrichment of monocytes-derived tumor-associated macrophages in the tumor microenvironment (TME) of DLBCL, which accelerated the tumor growth primarily by expressing cytokines such as IL-6, inducing angiogenesis, and exerting immunosuppressive effects through the expression of cytokines like IL-10; thus, conferring growth advantage to the ABC- DLBCL. To translate the findings into clinical application, we design a novel treatment strategy to overcome the poor outcomes in ABC-DLBCL patients with high IL-16 expression by combination of high-dose cyclophosphamide (CTX) and the bispecific antibody targeting CD20 and CD47 (IMM0306). IMM0306 effectively reversed the tumor-promoting effects of macrophages in subsets of ABC-DLBCL, while CTX induced IL-16 and increased the infiltration of macrophages in the TME. The increasing effector cells/tumor cells ratio further improved the antitumor effect of IMM0306. The combination of high-dose CTX and IMM0306 was confirmed to significantly inhibit the growth of ABC-DLBCL with high IL-16 expression. A clinical trial is being designed to evaluate the potential for clinical applications. Conclusion: In conclusion, this study provides insight into the expression and role of IL-16 in DLBCL. IL-16 is proposed to be highly expressed in ABC-DLBCL, and serum IL-16 indicate the tumor burden, treatment response, and clinical prognosis of DLBCL patients. IL-16 may be a key driver of the TME of DLBCL, which can confer growth advantage to the ABC-DLBCL. Our data provides a theoretical basis for future clinical treatment. A novel treatment regimen (IMM0306 and CTX) could attenuate the pro-oncogenic roles of IL-16 to prevent ABC-DLBCL progression.

Stratification of Multiple Myeloma Patients in the Commpass Study By Single Cell Transcriptomics of Plasma Cells Associates with Clinical Outcome

Introduction: There is substantial variation in the prognosis and response to therapy among multiple myeloma (MM) patients. This diversity is influenced by both intrinsic tumor features and the tumor microenvironment. We hypothesize that the clinical outcomes post IMiD and/or PI are linked to molecular subtypes dictated by tumor sample transcriptomics. We previously showed that molecular subtyping based on the CoMMpass bulk RNAseq data has an association with time to second-line therapy (log rank p = 0.0018; Hamidi et al., Blood 140: 7208-7210). Here, we applied the same molecular subtyping schema to single cell RNAseq isolated plasma cell transcriptomic profiles. Methods: Bone marrow biopsies from newly diagnosed MM patients (N = 200) in the MMRF CoMMpass study (NCT01454297) were collected, enriched by CD138 negative bead selection, and scRNAseq was performed by MMRF Immune Profiling Consortium, a collaboration between MMRF and laboratories at Beth Israel Deaconess Medical Center, Icahn School of Medicine at Mt. Sinai, Mayo Clinic, Washington University in St. Louis, and Emory University. After quality control and unsupervised clustering, plasma cells were subset out and pseudo-bulk expression profiles were generated for baseline samples with more than 100 plasma cells (N = 95). Unsupervised machine learning approach based on consensus non-negative matrix factorization (cNMF) was applied to identify the transcriptomics-based molecular subtypes. A random forest model, built from scRNAseq-based molecular subtypes (scNMF), was applied to bulk RNAseq data with a larger sample size (N = 754) to train a predictive molecular subtyping (pNMF). Potential cell-cell communication events based on scRNAseq were also predicted by LIgand-receptor ANalysis frAmework (LIANA). Results: 705,959 single cells passed the quality control and were broadly annotated as T/NK cells, B cells, plasma cells, myeloid cells, erythroid, progenitor, and stromal cells. Consistent with previous reports, CD138 negative bead selection did not completely remove plasma cells, resulting in an average of 18.5% plasma cells (n = 130,213). Subsequent unsupervised clustering of plasma cells exhibited a patient specific clustering pattern, suggestive of the heterogeneity of these tumor-derived cells. Unsupervised NMF on the pseudo-bulk expressions of baseline plasma cells identified five patient subsets. In the same cohort of patients (N = 95), subtypes assigned by the single cell had a better association with time to second-line therapy ( p-value = 0.0027 in scNMF as compared to p = 0.13 in bulk NMF). The median time to 2nd line of 2,443 days in scNMF5.1 versus 585 days in scNMF5.4; and the median time to 2nd line of 1,597 days in NMF5.2 versus 850 days in NMF5.3. A predicted subtyping based on scRNAseq data (pNMF) had a significantly better association with time to second-line therapy (Figure 1A, log rank p = 0.00088), compared to the published molecular subtyping based on bulk RNAseq (log rank p = 0.0018). The median time to 2nd line of 1,342 days in pNMF5.1 versus 597 days in pNMF5.5; compared to the median time to 2nd line of 1,347 days in NMF5.2 versus 850 days in NMF5.3. The best clinical outcome subset pNMF5.1 was characterized with higher levels of IFN pathways (Figure 1B, Hallmark interferon gamma and alpha pathways Wilcoxon p = 0.003 and p = 0.010, respectively) and higher levels of tumor-promoting inflammatory microenvironment (Hallmarks inflammatory response and TNFα signaling via NF-κB p = 0.003 and p = 0.010, respectively). The worst outcome subset pNMF5.5, on the other hand, displayed higher levels of glycolysis, a promoting factor of tumor development ( p < 0.001). Moreover, the total number of cell-cell interactions between the major cell types differed based on scRNAseq subtypes, informing the underlying biology driving the molecular heterogeneity. We identified a positive feedback loop of CXCL12/CXCR4 signaling between stromal and plasma cells, enhancing tumor cell proliferation. Conclusions: The identification of molecular subtypes based on single-cell data through an innovative unsupervised machine learning approach can have significant implications for determining which NDMM patients are likely to respond to established therapies; and the cellular level resolution allows deconvolution of cell-cell signaling, enhancing our understanding of the underlying molecular biology and mechanisms of resistance.

Paracrine activin B-NF-κB signaling shapes an inflammatory tumor microenvironment in gastric cancer via fibroblast reprogramming

BackgroundImportant roles of INHBB in various malignancies are increasingly identified. The underlying mechanisms in gastric cancer (GC) microenvironment are still greatly unexplored.MethodsThe clinical significance of INHBB and the correlation between INHBB and p-p65 in GC were assessed through analyzing publicly available databases and human paraffin embedded GC tissues. The biological crosstalk of INHBB between GC cells and fibroblasts was explored both in vitro and in vivo. RNA-seq analyses were performed to determine the mechanisms which regulating fibroblasts reprogramming. Luciferase reporter assay and chromatin immunoprecipitation (CHIP) assay were used to verify the binding relationship of p65 and INHBB in GC cells.ResultsOur study showed that INHBB level was significantly higher in GC, and that increased INHBB was associated with poor survival. INHBB positively regulates the proliferation, migration, and invasion of GC cells in vitro. Also, activin B promotes the occurrence of GC by reprogramming fibroblasts into cancer-associated fibroblasts (CAFs). The high expression of INHBB in GC cells activates the NF-κB pathway of normal gastric fibroblasts by secreting activin B, and promotes fibroblasts proliferation, migration, and invasion. In addition, activin B activates NF-κB pathway by controlling TRAF6 autoubiquitination to induce TAK1 phosphorylation in fibroblasts. Fibroblasts activated by activin B can induce the activation of p65 phosphorylation of GC cells by releasing pro-inflammatory factors IL-1β. p65 can directly bind to the INHBB promoter and increase the INHBB transcription of GC cells, thus establishing a positive regulatory feedback loop to promote the progression of GC.ConclusionsGC cells p65/INHBB/activin B and fibroblasts p65/IL-1β signal loop led to the formation of a whole tumor-promoting inflammatory microenvironment, which might be a promising therapeutic target for GC.

Amelioration of Tumor-promoting Microenvironment via Vascular Remodeling and CAF Suppression Using E7130: Biomarker Analysis by Multimodal Imaging Modalities.

E7130 is a novel anticancer agent created from total synthetic study of the natural compound norhalichondrin B. In addition to inhibiting microtubule dynamics, E7130 also ameliorates tumor-promoting aspects of the tumor microenvironment (TME) by suppressing cancer-associated fibroblasts (CAF) and promoting remodeling of tumor vasculature. Here, we demonstrate TME amelioration by E7130 using multi-imaging modalities, including multiplexed mass cytometry [cytometry by time-of-flight (CyTOF)] analysis, multiplex IHC analysis, and MRI. Experimental solid tumors characterized by large numbers of CAFs in TME were treated with E7130. E7130 suppressed LAP-TGFβ1 production, a precursor of TGFβ1, in CAFs but not in cancer cells; an effect that was accompanied by a reduction of circulating TGFβ1 in plasma. To our best knowledge, this is the first report to show a reduction of TGFβ1 production in TME. Furthermore, multiplex IHC analysis revealed reduced cellularity and increased TUNEL-positive apoptotic cells in E7130-treated xenografts. Increased microvessel density (MVD) and collagen IV (Col IV), an extracellular matrix (ECM) component associated with endothelial cells, were also observed in the TME, and plasma Col IV levels were also increased by E7130 treatment. MRI revealed increased accumulation of a contrast agent in xenografts. Moreover, diffusion-weighted MRI after E7130 treatment indicated reduction of tumor cellularity and interstitial fluid pressure. Overall, our findings strongly support the mechanism of action that E7130 alters the TME in therapeutically beneficial ways. Importantly, from a translational perspective, our data demonstrated MRI as a noninvasive biomarker to detect TME amelioration by E7130, supported by consistent changes in plasma biomarkers.

Dual Enzyme Cascade-Activated Popcorn-Like Nanoparticles Efficiently Remodeled Stellate Cells to Alleviate Pancreatic Desmoplasia.

In pancreatic cancer, excessive desmoplastic stroma severely impedes drug access to tumor cells. By reverting activated pancreatic stellate cells (PSCs) to quiescence, all-trans retinoic acid (ATRA) can attenuate their stromal synthesis and remodel the tumor-promoting microenvironment. However, its modulatory effects have been greatly weakened due to its limited delivery to PSCs. Therefore, we constructed a tripeptide RFC-modified gelatin/oleic acid nanoparticle (RNP@ATRA), which delivered ATRA in an enzyme-triggered popcorn-like manner and effectively resolved the delivery challenges. Specifically, surface RFC was cleaved by aminopeptidase N (APN) on the tumor endothelium to liberate l-arginine, generating nitric oxide (NO) for tumor-specific vasodilation. Then, massive nanoparticles were pushed from the vessels into tumors, showing 5.1- and 4.0-fold higher intratumoral accumulation than free ATRA and APN-inert nanoparticles, respectively. Subsequently, in the interstitium, matrix metalloproteinase-2-induced gelatin degradation caused RNP@ATRA to rapidly release ATRA, promoting its interstitial penetration and PSC delivery. Thus, activated PSCs were efficiently reverted to quiescence, and stroma secretion and vascular compression were reduced, thereby enhancing intratumoral delivery of small-molecule or nanosized chemotherapeutics. Ultimately, RNP@ATRA combined with chemotherapeutics markedly suppressed tumor growth and metastasis without causing additional toxicities. Overall, this work provides a potential nanoplatform for the efficient delivery of PSC-modifying agents in pancreatic cancer and other stroma-rich tumors.

Systemic Inflammatory Disorders, Immunosuppressive Treatment and Increase Risk of Head and Neck Cancers-A Narrative Review of Potential Physiopathological and Biological Mechanisms.

Head and neck cancers (HNCs) are known to present multiple factors likely to influence their development. This review aims to provide a comprehensive overview of the current scientific literature on the interplay between systemic inflammatory disorders, immunosuppressive treatments and their synergistic effect on HNC risk. Both cell-mediated and humoral-mediated systemic inflammatory disorders involve dysregulated immune responses and chronic inflammation and these inflammatory conditions have been associated with an increased risk of HNC development, primarily in the head and neck region. Likewise, the interaction between systemic inflammatory disorders and immunosuppressive treatments appears to amplify the risk of HNC development, as chronic inflammation fosters a tumor-promoting microenvironment, while immunosuppressive therapies further compromise immune surveillance and anti-tumor immune responses. Understanding the molecular and cellular mechanisms underlying this interaction is crucial for developing targeted prevention strategies and therapeutic interventions. Additionally, the emerging field of immunotherapy provides potential avenues for managing HNCs associated with systemic inflammatory disorders, but further research is needed to determine its efficacy and safety in this specific context. Future studies are warranted to elucidate the underlying mechanisms and optimize preventive strategies and therapeutic interventions.

Expression of Interleukin 23p19 in Colorectal Carcinoma: An Immunohistochemical Study with Clinico-Pathological Correlation

Background Cancer of the colon is the fourth most common cancer in the world, while cancer of the rectum is the eight most common. Together, colorectal carcinomas (CRCs) are the third most commonly diagnosed form of cancer globally, comprising about 11% of all cancer diagnoses. In Egypt, a rapid increase in CRC incidence has been observed with an unusually high rate under the age of 40. Interleukin 23 (IL-23) is a member of a family of pro-inflammatory cytokines, consisting of a p19 subunit and a p40 subunit. Studies showed that IL-23 signaling causes up-regulation of MMP9, increased angiogenesis, and reduced CD8+ T cell recruitment to tumors. Therefore, IL-23–mediated inflammatory processes might provide a tumor-promoting microenvironment. Objective To evaluate the immunohistochemical expression of IL23p19 in colorectal carcinoma with clinico-pathological correlation. Methods This is a retrospective study consisting of formalin fixed paraffin embedded tissue blocks of 25 cases diagnosed as colorectal carcinoma. All tissue blocks were retrieved from the archives of the pathology department at Al-Demerdash Hospital. Results There was a significant difference between CRC cases with different tumor stages as regard total IL23p19 expression, as 100% of cases with higher stage (T4a/b) had positive expression compared to 42.9% and 92.9% of T2 and T3 respectively. However, no significant difference was detected between cases with different tumor grades as regard total IL23p19 expression. Conclusion It can be concluded from our study that there is an association between IL23p19 expression and the pathological stage in CRC patients. IL23p19 could be used as a prognostic biomarker and a potential treatment target.

Dose-related immunomodulatory effects of recombinant TRAIL in the tumor immune microenvironment

BackgroundIn addition to specifically inducing tumor cell apoptosis, recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has also been reported to influence the cancer immune microenvironment; however, its underlying effects and mechanisms remain unclear. Investigating the immunomodulatory effects and mechanisms of recombinant TRAIL in the tumor microenvironment (TME) may provide an important perspective and facilitate the exploration of novel TRAIL strategies for tumor therapy.MethodsImmunocompetent mice with different tumors were treated with three doses of recombinant TRAIL, and then the tumors were collected for immunological detection and mechanistic investigation. Methodological approaches include flow cytometry analysis and single-cell sequencing.ResultsIn an immunocompetent mouse model, recombinant soluble mouse TRAIL (smTRAIL) had dose-related immunomodulatory effects. The optimal dose of smTRAIL (2 mg/kg) activated innate immune cells and CD8+ T cells, whereas higher doses of smTRAIL (8 mg/kg) promoted the formation of a tumor-promoting immune microenvironment to counteract the apoptotic effects on tumor cells. The higher doses of smTRAIL treatment promoted M2-like macrophage recruitment and polarization and increased the production of protumor inflammatory cytokines, such as IL-10, which deepened the suppression of natural killer (NK) cells and CD8+ T cells in the tumor microenvironment. By constructing an HU-HSC-NPG.GM3 humanized immune system mouse model, we further verified the immunomodulatory effects induced by recombinant soluble human TRAIL (shTRAIL) and found that combinational administration of shTRAIL and trabectedin, a macrophage-targeting drug, could remodel the tumor immune microenvironment, further enhance antitumor immunity, and strikingly improve antitumor effects.ConclusionOur results highlight the immunomodulatory role of recombinant TRAIL and suggest promising therapeutic strategies for clinical application.Graphical

PPARγ activation modulates the balance of peritoneal macrophage populations to suppress ovarian tumor growth and tumor-induced immunosuppression

BackgroundOvarian adenocarcinoma (OVAD) frequently metastasizes to the peritoneal cavity and manifests by the formation of ascites, which constitutes a tumor-promoting microenvironment. In the peritoneal cavity, two developmentally, phenotypically and functionally...

Spatial Heterogeneity in Cytoskeletal Mechanics Response to TGF-β1 and Hypoxia Mediates Partial Epithelial-to-Meshenchymal Transition in Epithelial Ovarian Cancer Cells.

Metastatic progression of epithelial ovarian cancer (EOC) involves the partial epithelial-to-mesenchymal transition (EMT) of cancer cells in the primary tumor and dissemination into peritoneal fluid. In part to the high degree of heterogeneity in EOC cells, the identification of EMT in highly epithelial cells in response to differences in matrix mechanics, growth factor signaling, and tissue hypoxia is very difficult. We analyzed different degrees of EMT by tracking changes in cell and nuclear morphology, along with the organization of cytoskeletal proteins. In our analysis, we see a small percentage of individual cells that show dramatic response to TGF-β1 and hypoxia treatment. We demonstrate that EOC cells are spatially aware of their surroundings, with a subpopulation of EOC cells at the periphery of a cell cluster in 2D environments exhibited a greater degree of EMT. These peripheral cancer cells underwent partial EMT, displaying a hybrid of mesenchymal and epithelial characteristics, which often included less cortical actin and more perinuclear cytokeratin expression. Collectively, these data show that tumor-promoting microenvironment conditions can mediate invasive cell behavior in a spatially regulated context in a small subpopulation of highly epithelial clustered cancer cells that maintain epithelial characteristics while also acquiring some mesenchymal traits through partial EMT.

EPEN-05. TRANSCRIPTIONAL FACTOR-DIRECTED EPIGENETIC REGULATION IN CANCER NEUROSCIENCE OF ZFTA-RELA EPENDYMOMA

Abstract ZFTA-RELA (ZRFUS) ependymoma is a pediatric brain tumor with dismal 10-year progression free survival rates. Prior studies revealed low somatic mutation load in pediatric ependymoma compared to adult glioma, suggesting that developmental mechanisms play a key role during disease development. Using human tissues and mouse tumors generated from our autochthonous in utero electroporation (IUE) model, we identified a cohort of developmental transcription factors (TFs) linked to ZRFUS by investigating histone serotonylation changes with other epigenetic datasets. We further performed in vivo barcode-screening and identified the ETS variant transcription factor 5 (ETV5) is sufficient and required for disease progression. To further reveal the disease mechanism driven by ETV5, we applied transcriptomic, epigenetic, and proteomic analyses, and found ETV5 suppresses neuropeptide Y (NPY) expression in mouse ZRFUS tumors by binding with epigenetic modulator, chromobox 3 (CBX3). We further found NPY overexpression delays tumor progression and suppressed tumor-driven neuronal activity in mice. This study suggests developmental TF can shape tumor-promoting microenvironment during disease progression through epigenetic regulation.