Tumor Niche Research Articles

Selenium Nanoparticles Show Anticancer Activity Through Regulation of HIF-1α and HIF-2α Under Hypoxic Condition in Liver Cancer Cells.

Tumor microenvironment has significant influence in therapeutic response and clinical outcome. Combination therapy is more effective in cancer treatment compared with monotherapy. Any chemical or drug that targets tumor microenvironment pathway, will be a boon to combination cancer chemotherapy. Combination therapy through micronutrient may have added advantage in clinical applications. Selenium (Se) is an essential micronutrient; Se in the form of Se nanoparticles (SeNPs) show efficient anticancer properties and may have the potential to target tumor niche such as hypoxic environment. The aim of this study was to find out the anticancer effect of SeNPs on cell line HepG2 under hypoxic condition and also to evaluate their effect on the translocation of hypoxia-inducible factors (HIFs) from cytoplasm to nucleus that help the cells to survive under hypoxic condition. It was found that the SeNPs induce HepG2 cell death in normoxic and hypoxic conditions, however, hypoxic condition showed higher LD50. SeNP concentration is directly proportional to cell death in both the conditions. Furthermore, intracellular accumulation of Se is not affected by hypoxia. SeNP-induced HepG2 cell death is due to increased DNA damage, nuclear condensation, and mitochondrial membrane potential disturbance. Furthermore, SeNPs were also found to decrease the translocation of HIFs from cytosol to the nucleus. After analyzing the results, it is concluded that SeNP treatment disturbs tumor niche through the inhibition of HIFs' translocation from cytosol to nucleus. SeNPs in synergy with primary drug, such as doxorubicin (DOX), may enhance the anticancer efficacy of DOX through regulation of HIFs, warranting further research.

Interferon-gamma is quintessential for NOS2 and COX2 expression in ER- breast tumors that lead to poor outcome

A strong correlation between NOS2 and COX2 tumor expression and poor clinical outcomes in ER breast cancer has been established. However, the mechanisms of tumor induction of these enzymes are unclear. Analysis of The Cancer Genome Atlas (TCGA) revealed correlations between NOS2 and COX2 expression and Th1 cytokines. Herein, single-cell RNAseq analysis of TNBC cells shows potent NOS2 and COX2 induction by IFNγ combined with IL1β or TNFα. Given that IFNγ is secreted by cytolytic lymphocytes, which improve clinical outcomes, this role of IFNγ presents a dichotomy. To explore this conundrum, tumor NOS2, COX2, and CD8+ T cells were spatially analyzed in aggressive ER–, TNBC, and HER2 + breast tumors. High expression and clustering of NOS2-expressing tumor cells occurred at the tumor/stroma interface in the presence of stroma-restricted CD8+ T cells. High expression and clustering of COX2-expressing tumor cells extended into immune desert regions in the tumor core where CD8+ T cell penetration was limited or absent. Moreover, high NOS2-expressing tumor cells were proximal to areas with increased satellitosis, suggestive of cell clusters with a higher metastatic potential. Further in vitro experiments revealed that IFNγ + IL1β/TNFα increased the elongation and migration of treated tumor cells. This spatial analysis of the tumor microenvironment provides important insight into distinct neighborhoods where stroma-restricted CD8+ T cells exist proximal to NOS2-expressing tumor niches that could have increased metastatic potential.

Centrosome amplification-related signature correlated with immune microenvironment and treatment response predicts prognosis and improves diagnosis of hepatocellular carcinoma by integrating machine learning and single-cell analyses.

Centrosome amplification is a well-recognized oncogenic driver of tumor initiation and progression across a variety of malignancies and has been linked with tumor aggressiveness, metastasis, and adverse prognosis. Nevertheless, the significance of centrosome amplification in HCC is not well understood. The TCGA dataset was downloaded for centrosome amplification-related signature construction using the LASSO-penalized Cox regression algorithm, while the ICGC dataset was obtained for signature validation. Single-cell RNA sequencing from GSE149614 was analyzed to profile gene expression and the liver tumor niche. A total of 134 centrosome amplification-related prognostic genes in HCC were detected and 6 key prognostic genes (SSX2IP, SPAG4, SAC3D1, NPM1, CSNK1D, and CEP55) among them were screened out to construct a signature with both high sensitivity and specificity in diagnosis and prognosis of HCC patients. The signature, as an independent factor, was associated with frequent recurrences, high mortality rates, advanced clinicopathologic features, and high vascular invasions. Moreover, the signature was intimately associated with cell cycle-related pathways and TP53 mutation profile, suggesting its underlying role in accelerating cell cycle progression and leading to liver cancer development. Meanwhile, the signature was also closely correlated with immunosuppressive cell infiltration and immune checkpoint expression, making it a vital immunosuppressive factor in the tumor microenvironment. Upon single-cell RNA sequencing, SSX2IP and SAC3D1 were found to be specially expressed in liver cancer stem-like cells, where they promoted cell cycle progression and hypoxia. This study provided a direct molecular link of centrosome amplification with clinical characteristics, tumor microenvironment, and clinical drug-response, highlighting the critical role of centrosome amplification in liver cancer development and therapy resistance, thereby providing valuable insights into prognostic prediction and therapeutic response of HCC.

Pericyte-Glioblastoma Cell Interaction: A Key Target to Prevent Glioblastoma Progression.

Multiple biological processes rely on direct intercellular interactions to regulate cell proliferation and migration in embryonic development and cancer processes. Tumor development and growth depends on close interactions between cancer cells and cells in the tumor microenvironment. During embryonic development, morphogenetic signals and direct cell contacts control cell proliferation, polarity, and morphogenesis. Cancer cells communicate with cells in the tumor niche through molecular signals and intercellular contacts, thereby modifying the vascular architecture and antitumor surveillance processes and consequently enabling tumor growth and survival. While looking for cell-to-cell signaling mechanisms that are common to both brain development and cancer progression, we have studied the infiltration process in glioblastoma multiforme (GBM), which is the most malignant primary brain tumor and with the worst prognosis. Cell-to-cell contacts, by means of filopodia-like structures, between GBM cells and brain pericytes (PCs) are necessary for adequate cell signaling during cancer infiltration; similarly, contacts between embryonic regions, via cytonemes, are required for embryo regionalization and development. This GBM-PC interaction provokes two important changes in the physiological function of these perivascular cells, namely, (i) vascular co-option with changes in cell contractility and vascular malformation, and (ii) changes in the PC transcriptome, modifying the microvesicles and protein secretome, which leads to the development of an immunosuppressive phenotype that promotes tumor immune tolerance. Moreover, the GTPase Cdc42 regulates cell polarity across organisms, from yeast to humans, playing a central role in GBM cell-PC interaction and maintaining vascular co-option. As such, a review of the molecular and cellular mechanisms underlying the development and maintenance of the physical interactions between cancer cells and PCs is of particular interest.

Dihydroartemisinin increased the abundance of Akkermansia muciniphila by YAP1 depression that sensitizes hepatocellular carcinoma to anti-PD-1 immunotherapy.

The effect of anti-programmed cell death 1 (anti-PD-1) immunotherapy is limited in patients with hepatocellular carcinoma (HCC). Yes-associated protein 1 (YAP1) expression increased in liver tumor cells in early HCC, and Akkermansia muciniphila abundance decreased in the colon. The response to anti-PD-1 treatment is associated with A. muciniphila abundance in many tumors. However, the interaction between A. muciniphila abundance and YAP1 expression remains unclear in HCC. Here, anti-PD-1 treatment decreased A. muciniphila abundance in the colon, but increased YAP1 expression in the tumor cells by mice with liver tumors in situ. Mechanistically, hepatocyte-specific Yap1 knockout (Yap1LKO) maintained bile acid homeostasis in the liver, resulting in an increased abundance of A. muciniphila in the colon. Yap1 knockout enhanced anti-PD-1 efficacy. Therefore, YAP1 inhibition is a potential target for increasing A. muciniphila abundance to promote anti-PD-1 efficacy in liver tumors. Dihydroartemisinin (DHA), acting as YAP1 inhibitor, increased A. muciniphila abundance to sensitize anti-PD-1 therapy. A. muciniphila by gavage increased the number and activation of CD8+ T cells in liver tumor niches during DHA treatment or combination with anti-PD-1. Our findings suggested that the combination anti-PD-1 with DHA is an effective strategy for liver tumor treatment.

Role of ionizing radiation activated NRF2 in lung cancer radioresistance

Radiotherapies are commonly used to target remaining tumor niches after surgery of solid tumors but are restricted due to therapeutic resistance. Several pathways of radioresistance have been reported in various cancers. This study investigates the pivotal role of Nuclear factor-erythroid 2–related factor 2 (NRF2) in the activation of DNA damage repair in lung cancer cells after x-rays exposure. To explore the NRF2 activation after ionizing irradiations, this study uses a knockdown of NRF2, which shows potential DNA damage after x-rays irradiation in lung cancers. This work further shows that NRF2 knockdown disrupts damaged DNA repair by inhibiting DNA-dependent protein kinase catalytic subunit. At the same time, NRF2 knockdown by shRNA considerably disparate homologous recombination by interfering with Rad51 expression. Further investigation of the associated pathway reveals that NRF2 activation mediates DNA damage response via the mitogen-activated protein kinase (MAPK) pathway as the knockout of NRF2 directly enhances intracellular MAPK phosphorylation. Similarly, both N-acetylcysteineand constitutive knockout of NRF2 disrupt DNA-dependent protein kinase catalytic subunit, while NRF2 knockout failed to upregulate Rad51 expression after irradiation in-vivo. Taken together, these findings advocate NRF2 plays a critical role in the development of radioresistance by upregulating DNA damage response via the MAPK pathway, which can be of great significance.

Multiomics and spatial mapping characterizes human CD8+ T cell states in cancer.

Clinically relevant immunological biomarkers that discriminate between diverse hypofunctional states of tumor-associated CD8+ T cells remain disputed. Using multiomics analysis of CD8+ T cell features across multiple patient cohorts and tumor types, we identified tumor niche-dependent exhausted and other types of hypofunctional CD8+ T cell states. CD8+ T cells in "supportive" niches, like melanoma or lung cancer, exhibited features of tumor reactivity-driven exhaustion (CD8+ TEX). These included a proficient effector memory phenotype, an expanded T cell receptor (TCR) repertoire linked to effector exhaustion signaling, and a cancer-relevant T cell-activating immunopeptidome composed of largely shared cancer antigens or neoantigens. In contrast, "nonsupportive" niches, like glioblastoma, were enriched for features of hypofunctionality distinct from canonical exhaustion. This included immature or insufficiently activated T cell states, high wound healing signatures, nonexpanded TCR repertoires linked to anti-inflammatory signaling, high T cell-recognizable self-epitopes, and an antiproliferative state linked to stress or prodeath responses. In situ spatial mapping of glioblastoma highlighted the prevalence of dysfunctional CD4+:CD8+ T cell interactions, whereas ex vivo single-cell secretome mapping of glioblastoma CD8+ T cells confirmed negligible effector functionality and a promyeloid, wound healing-like chemokine profile. Within immuno-oncology clinical trials, anti-programmed cell death protein 1 (PD-1) immunotherapy facilitated glioblastoma's tolerogenic disparities, whereas dendritic cell (DC) vaccines partly corrected them. Accordingly, recipients of a DC vaccine for glioblastoma had high effector memory CD8+ T cells and evidence of antigen-specific immunity. Collectively, we provide an atlas for assessing different CD8+ Tcell hypofunctional states in immunogenic versus nonimmunogenic cancers.

Abstract 5918: Interrogating host-intratumoral microbial interactions in pancreatic cancer

Abstract Cancer is increasingly becoming a rising cause of mortality worldwide. Microbiota, both within the gut and tumors, has emerged as a significant player influencing tumor growth and responses to therapies. The tumor niche provides a privileged microenvironment for microbial colonization. Recent evidence links microbiota and pancreatic tumorigenesis. Pancreatic ductal adenocarcinoma (PDAC), is an aggressive cancer surrounded by a highly immuno-suppressive tumor microenvironment (TME) which limits efficacy of most available therapies. We have previously reported that rare long-term survival in pancreatic cancer is associated with an intratumoral microbial signature which correlates with enhanced TME immunoactivation, suggestive of microbial mediated immune cell recruitment. A gut-to-tumor microbial crosstalk was also found in a human-to-mouse Fecal Microbiota Transplant (FMT) tumor model. While we have identified the vital role of microbiota in affecting tumor immunity, there is still a gap of knowledge about local microbial interactions within TME. Deeper understanding of the microbial mediated events that are triggered and follow PDAC development, which act to induce and support tumor growth, would provide us with potential novel targets that could be blocked to reverse PDAC immunosuppression and tumor growth. To interrogate the features of the pancreatic tumor microbial niche, we performed spatial co-detection of microbial and host targets to identify the cellular compartment which interacts with microbes inside the TME in clinical samples. We developed novel qualitative and quantitative imaging methodologies for evaluating crosstalk between the host and microbes. We also evaluated the effect of microbial burden on transcriptomic changes in host cells through multiple spatial sequencing approaches. Overall, we dissect the functional role of microbes in orchestrating TME organization and their effect on tumor signaling. Citation Format: Vidhi Chandra, Hajar Rajaei, Seyda Baydogan, Javier Gomez, Sammy Ferri-Borgogno, Anirban Maitra, Erick Riquelme, Ismet Sahin, Jared Burks, Michael Kim, Florencia McAllister. Interrogating host-intratumoral microbial interactions in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5918.

Abstract 184: Multiple Myeloma 3D model: A platform for testing drug effects on myeloma in conjunction with the bone marrow niche

Abstract Background: Multiple myeloma is a cancer that remains mostly incurable despite of the wide array of treatment options. In part, this may be due to its ability to develop resistance to therapy with time. To evaluate key aspects of the disease such as the protective effects of the tumor niche, the immune repertoire that could influence immunotherapies and the ability to study primary tumor cells, developing a new platform is necessary. In order to overcome the obstacles of growing primary myeloma cells and having a model that better replicates the tumor microenvironment, we used a 3D organoid model with patient-derived cells to evaluate different conditions to observe how current therapies affects cancer cells and the tumor niche in which they live. Methods: We used a 3D organoid model using patient bone marrow aspirates containing myeloma cells and stroma in a Matrigel scaffold and incubated over 21 days with partial media change containing key growth factors found in the bone marrow space to maintain cells alive. Established agents used to treat myeloma were used to expose the cultures and then monitored at different timepoints to study its immediate and delayed effect in the tumor cells, immune repertoire, and stroma. Bortezomib, Lenalidomide, Selinexor, and Dexamethasone, which are commonly used to treat myeloma patients and influence the immune compartment, were used at different concentrations. ATP, alamar blue, histology, and flow cytometry were performed at key timepoints of the experiment. Results: H&E sections demonstrated hematopoietic elements with good cellular preservation suggesting that the model may be a functional way to recapitulate the tumor environment. Patient-derived Multiple Myeloma organoids treated with Bortezomib 10nM and Selinexor 10uM and 30uM showed significant inhibition in viability compared to control. Lenalidomide 100 uM had variable effect in viability between organoids. Flow Cytometry analysis captured the immunomodulatory effect of Lenalidomide on the immune cells. Conclusions: The 3D organoid model was successful at maintaining viability for 21 days and having a heterogeneous representation of the tumor niche when using bone marrow aspirates of myeloma patients. The experiments performed allowed us to explore not just the effect of drugs on myeloma cells, but we can see the impact they have on the other cells in the bone marrow niche. A unique interest in the ability to evaluate changes in the immune compartment may help better understand immunotherapy and optimize personalized medicine by helping select the most effective treatments for a particular patient. Citation Format: Hanadi M. Rashad, Giovanni Insuasti, Graca Almeida-Porada, Cesar Rodriguez. Multiple Myeloma 3D model: A platform for testing drug effects on myeloma in conjunction with the bone marrow niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 184.

Abstract 4636: High-plex co-detection of RNA and protein to explore tumor-immune interactions utilizing RNAscope with Imaging Mass Cytometry

Abstract Purpose The next breakthroughs in immuno-oncology will be driven by high-plex tools that decipher the spatial arrangement of different cell types within the tumor microenvironment (TME). Imaging Mass Cytometry™ (IMC™) is a proven tool for the study of complex cellular interactions in the TME. It utilizes CyTOF® technology for simultaneous assessment of 40-plus protein markers at subcellular resolution without spectral overlap or background autofluorescence, thus providing unprecedented insight into the organization and function of the TME. Despite this, some protein targets are challenging to include in IMC as they have very few or no commercial antibodies available. Moreover, although cellular identity can easily be deciphered through detection of protein targets, knowledge of the cell’s transcriptome improves understanding of cellular function and activation state. Here, we present a robust and reliable workflow that combines the highly sensitive and specific RNAscope™ technology for RNA detection with the multiplexing capability of IMC to visualize key RNA and protein markers in the same tumor samples. Methods The RNAscope HiPlex v2 assay was combined with protein detection on the Hyperion+™ Imaging System to evaluate expression of both RNA and protein targets in formalin-fixed, paraffin-embedded (FFPE) tumor tissue microarray (TMA). The RNAscope assay was used with 12 target RNA marker probes and associated metal-labeled detection probes, suitable for use in IMC. The RNAscope HiPlex v2 assay workflow was followed as recommended up until applying the fluoros. Instead of using the fluoros, metal-conjugated probes were used for RNA detection. Metal-conjugated antibodies were used for protein detection in the same tissue. Results Target protein markers identified a range of immune cells, tumor cells, stromal cells, endothelial cells, and extracellular matrix. Co-detection of RNA and protein allowed visualization of several cytokines and chemokines, such as CXCL13, CXCL9, CXCL10, IFNγ, IL10, IL8, and IL1B, enabling the identification of the cellular source of these secreted factors. Additionally, immune cell subpopulations and their activation states were visualized using marker-specific antibodies. Immune cell hubs associated with anti-tumor immune responses were detected in tumor niches across the TMA. Conclusions Overall, combining RNAscope with protein co-detection on the IMC platform allowed simultaneous visualization of RNA and protein targets to interrogate the tumor microenvironment. The single-cell resolution offered by the RNAscope assay provides superior sensitivity for RNA detection in addition to the existing protein-detection capability of the IMC platform. This workflow can be applied to FFPE tissues to study therapeutic efficacy, stratify patients based on inflammatory signature, and study cell-cell interactions within the TME. Citation Format: James Pemberton, Smriti Kala, Anushka Dikshit, Clinton Hupple. High-plex co-detection of RNA and protein to explore tumor-immune interactions utilizing RNAscope with Imaging Mass Cytometry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4636.

L1CAM deployed perivascular tumor niche promotes vessel wall invasion of tumor thrombus and metastasis of renal cell carcinoma

The survival of tumor cells in the bloodstream, and vasculature adhesion at metastatic sites are crucial for tumor metastasis. Perivascular invasion aids tumor cell self-renewal, survival, and formation of metastases by facilitating readily available oxygen, nutrients, and endothelial-derived paracrine factors. Renal cell carcinoma (RCC) is among the most prevalent tumors of the urinary system, and the formation of venous tumor thrombus (VTT) is a characteristic feature of RCC. We observed high expression of L1CAM in the VTT with vessel wall invasion. L1CAM promotes the adhesion, migration, and invasion ability of RCC and enhances metastasis by interacting with ITGA5, which elicits activation of signaling downstream of integrin α5β1. L1CAM promotes ADAM17 transcription to facilitate transmembrane ectodomain cleavage and release of soluble L1CAM. In response to soluble L1CAM, vascular endothelial cells release several cytokines and chemokines. Endothelial-derived CXCL5 and its receptor CXCR2 promote the migration and intravasation of RCC toward endothelial cells suggesting that crosstalk between endothelial cells and tumor cells has a direct guiding role in driving the metastatic spread of RCC. LICAM plays a crucial role in the invasive ability of RCC, and regulation of L1CAM expression may contribute therapeutically to preventing RCC progression.

Abstract 2371: Stromal androgen signaling acts as tumor niches to drive prostatic basal epithelial progenitor initiated oncogenesis through IGFBP3 mediated IGF1 induced Wnt activation

Abstract Prostate cancer is the second most common cancer in men worldwide. The current treatment targeting androgen receptor (AR) expressing cells, androgen deprivation therapy (ADT), eventually fails in most prostate cancer patients who consequently develop castration resistant prostate cancer, an incurable disease. Although significant effort has been devoted to understanding the promotional role of AR action in prostate tumor cells, there is an urgent need to define the specific role of stromal AR in prostate tumorigenesis and needs to be evaluated for designing effective therapy.We directly examined AR in stromal Shh responsive Gli1 lineage cells in prostate oncogenesis and tumor development utilizing in vivo tissue recombination and novel genetically engineered mouse models. AR loss in stromal Gli1 lineage cells reduces prostate epithelial oncogenesis in xenograft models, combining transformed UGE by expression of stabilized β catenin with AR deficient or control UGM. A Myc induced prostatic epithelial tumor mouse model was combined with conditional deletion of AR in Gli1 lineage cells during prepubescent and adult age which showed significant impairment of prostate tumor development. Single cell RNA sequencing revealed a robust increased expression of insulin like growth factor binding protein 3 (IGFBP3) in AR deleted stromal Gli1 lineage cells. Interestingly although no gene manipulation occurred in the epithelium, basal epithelial cells showed reduced IGF1 induced Wnt signaling, due to the increased IGFBP3 expression. Through chromatin immunoprecipitation qPCR, we examined how deletion of AR in Gli1 lineage cells increases IGFBP3 expression. Stromal AR deletion directly alleviates the suppression of Sp1 regulating transcription of IGFBP3, leading to inhibition of oncogenic growth of tumor epithelium in a paracrine manner. To uncover the mechanism for stromal AR in Gli1 lineage as a tumor niche, we used organoid culture systems. In prostatic epithelial organoid cultures, addition of IGFBP3 or conditional media from AR deleted stromal cells blocked tumor cell growth. Overall, the data implicate the novel and specific role of stromal AR in Gli1 lineage cells as a tumor niche to support prostate epithelial tumorigenesis. Dysregulation of stromal AR on IGFBP3 IGF1 signaling suggest an underlying mechanism for hormone refractoriness through the IGF axes. Cotargeting reciprocal interactions of AR and IGF1 pathways between epithelial tumor cells and surrounding tumor niches may improve clinical outcomes for treating advanced prostate cancer. Therefore, given the importance of sex hormone, hedgehog, and IGF1 signaling pathways in human development and tumorigenesis raises new and relevant questions underlying androgen, Shh, and IGF regulated tumor cell niches in tumor initiation and progression. Citation Format: Alex Hiroto, Won Kyung Kim, Christian H. Nenninger, Alyssa J. Buckley, Zijie Sun. Stromal androgen signaling acts as tumor niches to drive prostatic basal epithelial progenitor initiated oncogenesis through IGFBP3 mediated IGF1 induced Wnt activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2371.

Abstract 3660: Bone mechanics influence cancer stem cell formation in osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary tumor of bone and is characterized by rapid bone remodeling accompanied by structural and mechanical heterogeneity across the tumor. Chemoresistance and recurrence of OS can be attributed to a subpopulation of OS cells known as cancer stem-like cells (OS-CSCs). It can be hypothesized that tumor niches possessing distinct structural and mechanical properties could be harboring different OS cell populations, and certain niches might be more conducive for OS-CSCs formation and maintenance. To evaluate the presence of OS-CSCs, mineralized collagen scaffolds mimicking heterogenous bone niches with distinct structural and mechanical properties were fabricated by changing collagen content and pore alignment and were cultured with the highly metastatic human OS cell line 143B. Scaffolds with 47% collagen by weight but different pore alignment were chosen for further analysis because they possessed different stiffness and topographical properties. Cytoskeletal changes, epigenetic changes, chemoresistance and chemoresistance-related pathways, and the levels of stemness genes were assessed in the 3D scaffold cultures. A consistent trend of OS-CSC phenotype was observed in the cell population grown in the softer bone niches (12 kPa) with aligned pores as compared to stiffer niche (31 kPa) with non-aligned pores. The cell population growing in softer, aligned scaffolds had a significantly higher expression of stemness genes (OCT4, NANOG, SOX2, ALDH1A1) and drug efflux gene ABCB1 and increased chemoresistance to doxorubicin and cisplatin. Epigenetic changes associated with cisplatin resistance in OS such as a decrease in methyl transferase EZH2 accompanied by the upregulation of LIF/Notch and PRKCA pathways was observed in cells grown in softer, aligned niches. Fluorescent imaging of actin organization showed rounder cells in the aligned niche indicative of the CSC phenotype, and stretched, elongated cells in the non-aligned niche indicative of a differentiated phenotype. Our study showed that the mechanical and structural properties of bone niches influence the formation of distinct OS cell populations. A softer niche with an aligned structure is more conducive for maintaining OS-CSCs. Citation Format: Zunaira Shoaib, Aleczandria S. Tiffany, Brendan Harley, Joseph Irudayaraj, Timothy M. Fan. Bone mechanics influence cancer stem cell formation in osteosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3660.

Biophysical cues of in vitro biomaterials-based artificial extracellular matrix guide cancer cell plasticity.

Clinical evidence supports a role for the extracellular matrix (ECM) in cancer plasticity across multiple tumor types. The lack of in vitro models that represent the native ECMs is a significant challenge for cancer research and drug discovery. Therefore, a major motivation for developing new tumor models is to create the artificial ECM in vitro. Engineered biomaterials can closely mimic the architectural and mechanical properties of ECM to investigate their specific effects on cancer progression, offering an alternative to animal models for the testing of cancer cell behaviors. In this review, we focused on the biomaterials from different sources applied in the fabrication of the artificial ECM and their biophysical cues to recapitulate key features of tumor niche. Furthermore, we summarized how the distinct biophysical cues guided cell behaviors of cancer plasticity, including morphology, epithelial-to-mesenchymal transition (EMT), enrichment of cancer stem cells (CSCs), proliferation, migration/invasion and drug resistance. We also discuss the future opportunities in using the artificial ECM for applications of tumorigenesis research and precision medicine, as well as provide useful messages of principles for designing suitable biomaterial scaffolds.

157 Identifying Spatial Transcriptomics Signaling Networks in Human Glioblastoma Using Graph-Based Machine Learning

INTRODUCTION: The median survival for glioblastoma multiforme (GBM) has only improved by 4.6 months in more than 100 years, underlying the need for innovative methods for understanding this devastating tumor. Spatial transcriptomics is a new method of whole-transcriptome assessment which allows for simultaneous visualization of mRNA expression in situ with histopathology. METHODS: We applied spARC to 25,517 Unique Molecular Identifiers from 10 surgically resected human IDH-mutant astrocytic gliomas (Grade II (n = 3) and Grade III (n = 3)) and GBMs (n = 4). spARC uses manifold learning, graph filters, data diffusion and diffusion filtration to enable discovery of gene-gene relationships, spatial co-localization of genes, and receptor-ligand interactions applicable across a variety of spatial transcriptomic methods. A Neuropathologist annotated whole-slide images blind to our computational analysis and we used in-situ hybridization and immunohistochemistry to demonstrate that spaRC imputation recovery of gene expression is superior to raw expression. RESULTS: We demonstrate that spARC can identify spatially restricted tumor niches of dense tumor, leading edge, infiltration/high tumor burden, hemoglobin, and vasculature with high specificity. These clusters can be identified with genes including CD44, VIM, LDHA (dense tumor); SNAP25, UCHL1, OLMF1 (leading edge); OLIG2, MBP, PDGFRA (Infiltrating/High Tumor Burden). In our assessment of ligand-receptor interactions we found that GBM samples were enriched for SPP1-CD44 signaling. We validated this using GBM TCGA data stratifying patients based on expression of SPP1 and CD44 genes and found that high expression conferred lower survival at 12 months. CONCLUSIONS: Spatial transcriptomics paired with graph-based machine learning methods applied to human GBMs is a powerful technique able to uncover signaling networks relevant to patient survival and may aid in developing new therapeutic strategies.

Exploring Monocytes-Macrophages in Immune Microenvironment of Glioblastoma for the Design of Novel Therapeutic Strategies

Gliomas are primary malignant brain tumors. These tumors seem to be more and more frequent, not only because of a true increase in their incidence, but also due to the increase in life expectancy of the general population. Among gliomas, malignant gliomas and more specifically glioblastomas (GBM) are a challenge in their diagnosis and treatment. There are few effective therapies for these tumors, and patients with GBM fare poorly, even after aggressive surgery, chemotherapy, and radiation. Over the last decade, it is now appreciated that these tumors are composed of numerous distinct tumoral and non-tumoral cell populations, which could each influence the overall tumor biology and response to therapies. Monocytes have been proved to actively participate in tumor growth, giving rise to the support of tumor-associated macrophages (TAMs). In GBM, TAMs represent up to one half of the tumor mass cells, including both infiltrating macrophages and resident brain microglia. Infiltrating macrophages/monocytes constituted ~ 85% of the total TAM population, they have immune functions, and they can release a wide array of growth factors and cytokines in response to those factors produced by tumor and non-tumor cells from the tumor microenvironment (TME). A brief review of the literature shows that this cell population has been increasingly studied in GBM TME to understand its role in tumor progression and therapeutic resistance. Through the knowledge of its biology and protumoral function, the development of therapeutic strategies that employ their recruitment as well as the modulation of their immunological phenotype, and even the eradication of the cell population, can be harnessed for therapeutic benefit. This revision aims to summarize GBM TME and localization in tumor niches with special focus on TAM population, its origin and functions in tumor progression and resistance to conventional and experimental GBM treatments. Moreover, recent advances on the development of TAM cell targeting and new cellular therapeutic strategies based on monocyte/macrophages recruitment to eradicate GBM are discussed as complementary therapeutics.

A digital nanoplasmonic microarray immunosensor for multiplexed cytokine monitoring during CAR T-cell therapy from a leukemia tumor microenvironment model

The release of cytokines by chimeric antigen receptor (CAR) T-cells and tumor resident immune cells defines a significant part of CAR T-cell functional activity and patient immune responses during CAR T-cell therapy. However, few studies have so far precisely characterized the cytokine secretion dynamics in the tumor niche during CAR T-cell therapy, which requires multiplexed, and timely biosensing platforms and integration with biomimetic tumor microenvironment. Herein, we implemented a digital nanoplasmonic microarray immunosensor with a microfluidic biomimetic Leukemia-on-a-Chip model to monitor cytokine secretion dynamics during CD19 CAR T-cell therapy against precursor B-cell acute lymphocytic leukemia (B-ALL). The integrated nanoplasmonic biosensors achieved precise multiplexed cytokine measurements with low operating sample volume, short assay time, heightened sensitivity, and negligible sensor crosstalk. Using the digital nanoplasmonic biosensing approach, we measured the concentrations of six cytokines (TNF-α, IFN-γ, MCP-1, GM-CSF, IL-1β, and IL-6) during first 5 days of CAR T-cell treatment in the microfluidic Leukemia-on-a-Chip model. Our results revealed a heterogeneous secretion profile of various cytokines during CAR T-cell therapy and confirmed a correlation between the cytokine secretion profile and the CAR T-cell cytotoxic activity. The capability to monitor immune cell cytokine secretion dynamics in a biomimetic tumor microenvironment could further help in study of cytokine release syndrome during CAR T-cell therapy and in development of more efficient and safer immunotherapies.

Myeloid cells from Langerhans cell histiocytosis patients exhibit increased vesicle trafficking and an altered secretome capable of activating NK cells.

Langerhans cell histiocytosis (LCH) is a potentially life-threatening inflammatory myeloid neoplasia linked to pediatric neurodegeneration, whereby transformed LCH cells form agglomerated lesions in various organs. Although MAP-kinase pathway mutations have been identified in LCH cells, the functional consequences of these mutations and the mechanisms that cause the pathogenic behavior of LCH cells are not well understood. In our study, we used an in vitro differentiation system and RNA-sequencing to compare monocyte-derived dendritic cells from LCH patients to those derived from healthy controls or patients with Crohn's disease, a non-histiocytic inflammatory disease. We observed that interferon-γ treatment exacerbated intrinsic differences between LCH patient and control cells, including strikingly increased endo- and exocytosis gene activity in LCH patients. We validated these transcriptional patterns in lesions and functionally confirmed that LCH cells exhibited increased endo- and exocytosis. Furthermore, RNA-sequencing of extracellular vesicles revealed the enrichment of pathological transcripts involved in cell adhesion, MAP-kinase pathway, vesicle trafficking and T-cell activation in LCH patients. Thus, we tested the effect of the LCH secretome on lymphocyte activity and found significant activation of NK cells. These findings implicate extracellular vesicles in the pathology of LCH for the first time, in line with their established roles in the formation of various other tumor niches. Thus, we describe novel traits of LCH patient cells and suggest a pathogenic mechanism of potential therapeutic and diagnostic importance.

Immunomodulatory function and anti-tumor mechanism of natural polysaccharides: A review.

Polysaccharides extracted from natural resources have attracted extensive attention in biomedical research and pharmaceutical fields, due to their medical values in anti-tumor, immunomodulation, drug delivery, and many other aspects. At present, a variety of natural polysaccharides have been developed as adjuvant drugs in clinical application. Benefit from their structural variability, polysaccharides have great potential in regulating cellular signals. Some polysaccharides exert direct anti-tumor effects by inducing cell cycle arrest and apoptosis, while the majority of polysaccharides can regulate the host immune system and indirectly inhibit tumors by activating either non-specific or specific immune responses. As the essential of microenvironment in the process of tumor development has been gradually revealed, some polysaccharides were found to inhibit the proliferation and metastasis of tumor cells via tumoral niche modulation. Here, we focused on natural polysaccharides with biomedical application potential, reviewed the recent advancement in their immunomodulation function and highlighted the importance of their signaling transduction feature for the antitumor drug development.

Tumor-associated macrophages induce inflammation and drug resistance in a mechanically tunable engineered model of osteosarcoma

The tumor microenvironment is a complex and dynamic ecosystem composed of various physical cues and biochemical signals that facilitate cancer progression, and tumor-associated macrophages are especially of interest as a treatable target due to their diverse pro-tumorigenic functions. Engineered three-dimensional models of tumors more effectively mimic the tumor microenvironment than monolayer cultures and can serve as a platform for investigating specific aspects of tumor biology within a controlled setting. To study the combinatorial effects of tumor-associated macrophages and microenvironment mechanical properties on osteosarcoma, we co-cultured human osteosarcoma cells with macrophages within biomaterials-based bone tumor niches with tunable stiffness. In the first 24 h of direct interaction between the two cell types, macrophages induced an inflammatory environment consisting of high concentrations of tumor necrosis factor alpha (TNFα) and interleukin (IL)-6 within moderately stiff scaffolds. Expression of Yes-associated protein (YAP), but not its homolog, transcriptional activator with PDZ-binding motif (TAZ), in osteosarcoma cells was significantly higher than in macrophages, and co-culture of the two cells slightly upregulated YAP in both cells, although not to a significant degree. Resistance to doxorubicin treatment in osteosarcoma cells was correlated with inflammation in the microenvironment, and signal transducer and activator of transcription 3 (STAT3) inhibition diminished the inflammation-related differences in drug resistance but ultimately did not improve the efficacy of doxorubicin. This work highlights that the biochemical cues conferred by tumor-associated macrophages in osteosarcoma are highly variable, and signals derived from the immune system should be considered in the development and testing of novel drugs for cancer.