Tumor-conditioned Medium Research Articles

Sirtinol, a SIRT1 inhibitor, inhibits the EMT and metastasis of 4T1 breast cancer cells and impacts the tumor microenvironment.

The impact of epigenetic drugs on metastasis and the immunological microenvironment is poorly understood. In this study, we looked at how sirtinol, a SIRT1 inhibitor, affected epithelial-mesenchymal transition (EMT), metastasis, and the immune cells. In vitro experiments were carried out using tumor conditioned medium (TCM). For in vivo experiments, sirtinol was administered i.p. in tumor bearing BALB/c mice at a dose of 2 mg/kg body weight either alone or in combination with cisplatin. Estimation of cytokines was carried out using ELISA or ELIspot. Estimation of different markers was done using flow cytometry or western blot. Sirtinol, a SIRT1 inhibitor, was found to be cytotoxic to 4T1 breast cancer cells with no synergistic effects with cisplatin, both under in vitro and in vivo conditions (p < 0.05). Sirtinol significantly reduced cancer cell metastasis to the spleen which was supported by in vitro findings such as decreased vimentin expression and cell mobility in migration and wound healing assays (p < 0.01). Studies on the effects of 4T1 tumor-conditioned medium on spleen cells indicated changes in T cell proliferation as well as differentiation (p < 0.01). In tumor bearing mice, spleen cells showed elevated IFN-γ secretion, increased CD11b+ cells, and decreased T cells (p < 0.01). This was reversed by sirtinol as well as the combination treatment, which may also have contributed to metastasis inhibition (p < 0.01). Sirtinol, a SIRT1 inhibitor inhibits EMT and metastasis of 4T1 breast cancer cells and also has an impact on the immune microenvironment.

8636 Characterization Of The Role Of Neutrophils In Promoting Progression Of Lymphangioleiomyomatosis

Abstract Disclosure: T.R. Henson: None. B. Minor: None. S.R. Hammes: None. Lymphangioleiomyomatosis (LAM) is a rare, multisystemic disease that is associated with cystic lung destruction. LAM is caused by small smooth muscle cell-like tumors throughout the lung that contain deleterious mutations in the tuberous sclerosis complex genes TSC1 or TSC2. This attenuates the activity of the mammalian target of rapamycin complex 1 (mTORC1) causing increased proliferation of the smooth muscle cells. LAM is a sexually dimorphic disease that primarily affects genetic females. Clinical observations implicate the role of estrogen in disease progression, as it is exacerbated during pregnancy and by use of exogenous estrogens in birth control. LAM tumor-derived cells lines have shown that direct estrogen stimulation of cell proliferation is modest compared to the dramatic in vivo effects of estradiol (E2) stimulation in a uterine-specific TSC2-null mouse model. Creation of a monoclonal cell line from these mice resulted in the generation of the LTM3 cell line, which also demonstrates low response to direct estrogen stimulation in vitro. This indicates that direct E2 stimulation may act upon other cell types in the tumor microenvironment. Immunophenotyping of uterine-specific TSC2-null mice has shown that the tumor microenvironment is enriched with myeloid cells specifically neutrophils. Functional analysis of neutrophil suppression in vivo has shown a reduction in myometrial growth in the uterine-specific TSC2-null mice, indicating a key role for neutrophil-mediated LAM tumor growth. Altered transcriptional regulation of neutrophils further highlights the importance of neutrophils in LAM progression. Bulk messenger RNA sequencing revealed E2 dependent increase in the neutrophil derived serine protease neutrophil elastase (NE). NE in vitro has been shown to increase migration, invasion, and proliferation of LAM cell lines, while inhibition in vivo has shown a reduction to the myometrial growth. Previously published data by the lab has shown that E2 increases tumor induced expansion of neutrophils in the bone marrow. Isolation and purification of Neutrophils from mouse bone marrow stimulated with tumor conditioned media from the LTM3 cells has shown signs of activation via upregulated gene expression. The focus of the current research has been the characterization of bone marrow derived neutrophils. Currently working with an in vitro system to study the proliferation of LTM3 and the rat leiomyoma (ELT3) cell lines when they are co-cultured with neutrophils. Understanding the mechanism by which neutrophils are activated and interact with LAM cells can give understanding to disease progression may provide insight into future treatment options. Presentation: 6/3/2024

The Effects of Hypoxia on the Immune-Metabolic Interplay in Liver Cancer.

M2-like macrophages promote tumor growth and cancer immune evasion. This study used an in vitro model to investigate how hypoxia and tumor metabolism affect macrophage polarization. Liver cancer cells (HepG2 and VX2) and macrophages (THP1) were cultured under hypoxic (0.1% O2) and normoxic (21% O2) conditions with varying glucose levels (2 g/L or 4.5 g/L). Viability assays and extracellular pH (pHe) measurements were conducted over 96 hours. Macrophages were exposed to the tumor-conditioned medium (TCM) from the cancer cells, and polarization was assessed using arginase and nitrite assays. GC-MS-based metabolic profiling quantified TCM meta-bolites and correlated them with M2 polarization. The results showed that pHe in TCMs decreased more under hypoxia than normoxia (p < 0.0001), independent of glucose levels. The arginase assay showed hypoxia significantly induced the M2 polarization of macrophages (control group: p = 0.0120,0.1%VX2-TCM group: p = 0.0149, 0.1%HepG2-TCM group: p < 0.0001, 0.1%VX2-TCMHG group: p = 0.0001, and 0.1%HepG2-TCMHG group: p < 0.0001). TCMs also induced M2 polarization under normoxic conditions, but the strongest M2 polarization occurred when both tumor cells and macrophages were incubated under hypoxia with high glucose levels. Metabolomics revealed that several metabolites, particularly lactate, were correlated with hypoxia and M2 polarization. Under normoxia, elevated 2-amino-butanoic acid (2A-BA) strongly correlated with M2 polarization. These findings suggest that targeting tumor hypoxia could mitigate immune evasion in liver tumors. Lactate drives acidity in hypoxic tumors, while 2A-BA could be a therapeutic target for overcoming immunosuppression in normoxic conditions.

Non-small cell lung cancer cells and concomitant cancer therapy induce a resistance-promoting phenotype of tumor-associated mesenchymal stem cells.

The tumor microenvironment gained attraction over the last decades as stromal cells significantly impact on tumor development, progression and metastasis, and immune evasion as well as on cancer therapy resistance. We previously reported that lung-resident mesenchymal stem cells (MSCs) were mobilized and activated in non-small cell lung cancer (NSCLC) progression and could even mediate radiation resistance in co-cultured NSCLC cells. We investigated how MSCs were affected by NSCLC cells in combination with cancer (radiation) therapy in indirect co-cultures using tumor-conditioned medium and Transwells or direct three-dimensional NSCLC-MSC spheroid co-cultures in order to unravel the resistance-mediating action of tumor-associated MSCs. Although no obvious phenotypic and functional alterations in MSCs following NSCLC co-culture could be observed, MSC senescence was induced following co-applied radiotherapy (RT). Global gene expression profiling, in combination with gene set enrichment analysis upon treatment, was used to confirm the senescent phenotype of irradiated MSC and to reveal relevant senescence-associated secretory phenotype (SASP) factors that could meditate NSCLC RT resistance. We identified senescent tumor-associated MSC-derived serine proteinase inhibitor (serpin) E1/PAI1 as potential SASP factor mediating NSCLC progression and RT resistance. Specified intra-tumor-stroma interactions and cell type-specific pro-tumorigenic functions could not only improve lung cancer classification but could even be used for a more precise profiling of individual patients, finally paving an additional way for the discovery of potential drug targets for NSCLC patients.

DIPG-51. DIFFUSE MIDLINE GLIOMAS HIJACK MYELOID CELL POPULATIONS TO INITIATE AN ANTI-INFLAMMATORY TUMOR IMMUNE MICROENVIRONMENT

Abstract BACKGROUND Diffuse midline gliomas (DMG) are universally fatal pediatric brain tumors, for which no effective treatment exists. These tumors are known for their “cold” non-inflammatory tumor-immune microenvironment (TIME), which is thought to be a major hurdle for immunotherapy efficacy. METHODS/RESULTS To further investigate the mechanisms that drive this cold environment, we allografted immunocompetent mice with Histone 3.3 Lysine 27 (H3.3K27M) and 3.1 Lysine 27 (H3.1K27M) mutant murine DMG tumors, created through intra uterine electroporation, and performed single-cell analyses on the tumors that developed. Using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITEseq) we mapped the single-cell RNA and protein expression profiles of the TIME of these tumors and healthy pontine regions, with a dominant focus on the myeloid cell populations. Through differential expression and pathway enrichment analyses, we found that DMG-microglia populations upregulate anti-inflammatory pathways and, over time, are pushed out by infiltrating anti-inflammatory macrophages. We confirmed these findings in single-cell sequencing data from DMG patients, taken at diagnosis and at late stage disease. Next, we established microglia cultures in vitro, by differentiating monocytes from healthy donors into microglia. We discovered that treatment of these microglia with tumor-conditioned medium, taken from primary DMG cultures, diminished their pro-inflammatory potential after stimulation with lipopolysaccharides (LPS), as observed by cytokine release arrays that showed a strong downregulation of IL-6 and TNF-alpha release, amongst others. Subsequently, using a Seahorse metabolic analyzer, we found that glycoATP potential was diminished in DMG-primed microglia after LPS stimulation, further suggesting that DMG-excreted factors are responsible for blocking the pro-inflammatory potential of microglia. CONCLUSIONS Taken together, these findings increase our understanding on how DMG cells reprogram tumor-associated myeloid cells to generate an immune-cold TIME, which will aid the scientific community to find therapeutic solutions to reverse this phenomenon and improve the efficacy of immunotherapy in these devastating malignancies.

Syngeneic mesenchymal stem cells loaded with telomerase-dependent oncolytic adenoviruses enhance anti-metastatic efficacy.

Oncolytic adenoviruses have emerged as a promising therapeutic approach for cancer therapy. However, systemic delivery of the viruses to metastatic tumors remains a major challenge. Mesenchymal stem cells (MSCs) possess tumor tropism property and can be used as cellular vehicles for delivering oncolytic adenoviruses to tumor sites. Since telomerase activity is found in ~90% of human carcinomas, but undetected in normal adult cells, the human telomerase reverse transcriptase gene (TERT) promoter can be exploited for regulating the replication of oncolytic adenoviruses. Here, we evaluated the antitumor effects of syngeneic murine MSCs loaded with the luciferase-expressing, telomerase-dependent oncolytic adenovirus Ad.GS2 (MSC-Ad.GS2) and Ad.GS2 alone on metastatic MBT-2 bladder tumors. MSCs supported a low degree of Ad.GS2 replication, which could be augmented by coculture with MBT-2 cells or tumor-conditioned medium (TCM), suggesting that viral replication is increased when MSC-Ad.GS2 migrates to tumor sites. MBT-2 cells and TCM enhanced viral replication in Ad.GS2-infected MSCs. SDF-1 is a stem cell homing factor. Our results suggest that the SDF-1/STAT3/TERT signaling axis in MSCs in response to the tumor microenvironment may contribute to the enhanced replication of Ad.GS2 carried by MSCs. Notably, we demonstrate the potent therapeutic efficacy of systemically delivered MSC-Ad.GS2 in pleural disseminated tumor and experimental metastasis models using intrapleural and tail vein injection of MBT-2 cells, respectively. Treatment with MSC-Ad.GS2 significantly reduced tumor growth and prolonged the survival of mice bearing metastatic bladder tumors. Since telomerase is expressed in a broad spectrum of cancers, this therapeutic strategy may be broadly applicable.

Cancer Cell Small Molecule Secretome Induces the Immune Checkpoint NKG2A and Dysfunction of Human CD8+ T Cells.

PD-1 blockade has been approved for head and neck squamous cell carcinoma (HNSCC) patients. However, many HNSCC patients do not respond to this treatment, and other tumor microenvironmental factors may promote resistance to PD-1 blockade. We previously identified increased expression of the inhibitory receptor NKG2A on CD8+ T cells in HNSCC tumors compared with T cells in matching PBMC samples. Mechanisms that promote NKG2A expression and the role of NKG2A on human T cells in the tumor microenvironment, however, are uncertain. In this study, we show that tumor-conditioned media (TCM) of HNSCC cancer cell lines or ascites fluid from colorectal carcinoma patients is sufficient to induce the expression of NKG2A and other inhibitory receptors on activated CD8+ T cells isolated from PBMCs of healthy donors. Boiling or small molecular mass cutoff filtering did not eliminate the effect of TCM, suggesting that a small molecule promotes NKG2A. T cell activation in TCM decreased the basal and maximal mitochondrial respiration to metabolically restrain CD8+ T cells. Functionally, T cell activation in TCM reduced CD8+ T cell cytotoxicity as shown by lower production of cytokines, granzyme B, and perforin. Furthermore, TCM prevented CD8+ T cells from killing cancer cells in response to an anti-CD19/anti-CD3 bispecific T cell engager. Thus, a small secreted molecule from HNSCC cells can induce NKG2A expression and promote T cell dysfunction. Our findings may lead to targets for novel cancer therapies or biomarkers for NKG2A blockade response and provide a model to study T cell dysfunction and impaired metabolism.

Abstract PO5-25-05: Crosstalk between adipocyte and tumor cells with ESR1 mutations via adipsin enhances tumor growth

Abstract While endocrine therapies have shown clinical benefits for breast cancer with ESR1 mutations, resistance development is a significant challenge. Evidence suggests that the complement system is critical to tumor development, and recent studies highlight the role of adipsin in breast cancer growth. In a previous study, we characterized the role of complement pathway-mediated adipsin in ESR1 mutant cell lines (Y537S and D538G), identifying the upregulation of adipsin, C3, and C3aR in ESR1 mutant cells. The interaction between C3a and C3aR enhanced ESR1 mutant cell growth, and inhibiting C3aR abolished cell growth and increased apoptosis. Combining C3aR inhibitors with fulvestrant or CDK4/6 inhibitors had a synergistic inhibitory effect on ESR1 mutant cells. Our findings suggest that the complement pathway plays a role in ESR1 mutant cell proliferation and that C3aR is a potential target to mitigate ESR1 mutant breast cancer growth and metastasis. Additionally, we demonstrated the critical role of the feedback loop of adipsin in ESR1 mutant cell growth in adipocyte crosstalk. Using differentiated mouse 3T3-L1 adipocytes induced with tumor-conditioned media from ESR1 mutant cells, we observed the upregulation of adipsin, C3, and C3a in the adipocyte secretome, which enhanced C3a levels. Blocking the interaction between C3a and C3aR in ESR1 mutant cells using a C3aR inhibitor significantly inhibited ESR1 mutant cell growth compared to wild-type cells and enhanced apoptosis. Our findings suggest that upregulation of adipsin from ESR1 mutant cells and adipocytes in crosstalk increases tumor cell growth. Future studies will investigate the critical role of adipsin's auto- and paracrine effects in breast cancer with ESR1 mutations. Citation Format: Artem Belyakov, Kideok Jin. Crosstalk between adipocyte and tumor cells with ESR1 mutations via adipsin enhances tumor growth [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 PO5-25-05.

Overexpression of SOCS2 Inhibits EMT and M2 Macrophage Polarization in Cervical Cancer via IL-6/JAK2/STAT3 Pathway.

SOCS2 is a member of the suppressor of cytokine signaling (SOCS) protein family associated with the occurrence and development of multiple cancers. This study revealed the expression and molecular mechanisms of SOCS2 in cervical cancer. In this study, RT-qPCR, Western Blot, and immunohistochemistry were used to detect the expression level of SOCS2 in cervical cancer tissues and tumor cells. We overexpressed SOCS2 in SiHa cells via lentivirus. In-vitro experiments were used to investigate the changes in cervical cancer cell proliferation, migration, and invasion ability before and after SOCS2 overexpression. Western Blot was used to detect the expression of IL-6/JAK2/STAT3 pathway and EMTrelated proteins. M0 macrophages were co-cultured with the tumor-conditioned medium. The effect of SOCS2 on macrophage polarization was examined by RT-qPCR. SOCS2 expression level was significantly downregulated in cervical cancer tissues. SOCS2 was negatively correlated with CD163+M2 macrophages. Overexpression of SOCS2 inhibited the proliferation, migration, and invasion of cervical cancer cells. The expressions of Twist- 2, N-cadherin, and Vimentin were decreased, while the expression of E-cadherin was increased. Moreover, the expression of IL-6, p-JAK2, and p-STAT3 were decreased. After the addition of RhIL-6, the expression of E-cadherin protein in the LV-SOCS2 group was reversed. CM in the LV-SOCS2 group inhibited the polarization of M2 macrophages. SOCS2 acts as a novel biological target and suppressor of cervical cancer through IL- 6/JAK2/STAT3 pathway.

Abstract 2782: Characterization of quiescence-associated secretory profile (QuASP) in cancer: A novel defining feature and therapeutic target

Abstract Senescent cells have a unique senescence-associated secretory profile (SASP) and understanding SASP has contributed to numerous advances in knowledge on aging and cancer biology. Quiescence, which is an important yet poorly understood mechanism for chemoresistance, is related but distinct from senescence. Quiescence shares a non-proliferative feature with senescence but is a reversible state in which quiescent cells can reenter the cell cycle. We hypothesized that, analogous to senescent cells, quiescent cells have a distinct quiescence-associated secretory profile (QuASP) and that defining the QuASP will uncover new genes and pathways associated with cancer cell viability and therapeutic resistance. Indeed, suggesting quiescent cells have a unique and biologically important secretome, we found that tumor conditioned medium (TCM) from quiescent ovarian cancer cells (qOvCa) vs. TCM from proliferating cancer cells increased chemoresistance. Similarly, TCM from quiescent cancer cells vs. TCM from proliferating cancer cells suppressed T cell proliferation and INF-gamma/Granzyme B secretion, suggesting a QuASP immunosuppressive effect. RNASeq analysis of qOvCa cells revealed the secreted factor Follistatin (FST) is upregulated in qOvCa and acted as a paracrine pattern to induce chemoresistance. Furthermore, FST was found to be upregulated in peritumoral fluid following chemotherapy and FST levels in patients with OvCa predicted poor prognosis. To identify additional QuASP factors, we used an ER-BIOID labeled system and LC-MS/MS to characterize the secreted proteins from qOvCa induced by serum starvation compared with normal cells in three OvCa cell lines (PT412, PT340, HEY1). Quiescent OvCa cells indeed have a secretome that is highly unique from proliferating cells, with 137/577 upregulated proteins (LFC&amp;gt;1) and 65/577 downregulated proteins (LFC←1 or no detection in quiescence). Several proteins identified as upregulated have been linked to immunosuppression and/or chemoresistance, oncogenesis and cell cycle were selected for validation and further analysis (CLU, VGF, SERPINE1). In conclusion, we find quiescent cells have a unique quiescence associated secretory profile and that the QuASP induces chemoresistance and immunosuppression. As such, this study has important implications for cancer therapy. Citation Format: Qi Jiang, Santiago Panesso-Gómez, Divya Natesan, Matthew L. MacDonald, Ronald J. Buckanovich. Characterization of quiescence-associated secretory profile (QuASP) in cancer: A novel defining feature and therapeutic target [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 2782.

Abstract 45: Driving AMPK signaling in CD4+ CART cells enhances metabolic capacity, memory formation, and anti-leukemia activity

Abstract Background: While Chimeric Antigen Receptor (CAR)T cell therapy has seen great success in pediatric and young adult acute lymphoblastic leukemia, up to 50% of patients will relapse following treatment. Increased memory formation and mitochondrial capacity, particularly in the CD4+ subset, have been linked to improved CART cell function and enhanced in vivo persistence, leading to long-term relapse-free survival. AMP-activated protein kinase (AMPK) is a cellular energy sensor responsible for promoting metabolic efficiency and T cell memory formation. We have previously demonstrated AMPK activity can be increased in human T cells via overexpression of the AMPKγ2 domain. We hypothesized that overexpression of AMPKγ2 in CART cells would enhance their metabolic capacity and improve their anti-leukemia function. Methods: Human CART cells bearing a CD19-CAR utilizing a 41BB costimulatory domain were generated from healthy human donors via lentiviral transduction and co-transduced with AMPKγ2 (AMPK) or an empty vector (EV) control. Flow sorting for the co-transduced populations yielded &amp;gt;95% CD4+ CART cells. CARTs were expanded in IL-2 for approximately 2 weeks, followed by overnight stimulation and subsequent metabolic analysis using the Seahorse Metabolic Analyzer. Memory status was assessed by CD62L and CCR7 co-expression. In vitro CART function was assessed using Zs-green+ NALM6 targets, with loss of green fluorescence over time measured on the Incucyte analyzer. Where indicated, tumor conditioned media was harvested from 72-hour NALM6 cultures. For in vivo experiments, immunodeficient NOD-scid IL2Rγnull (NSG) mice were injected with luciferase+ NALM6 targets, followed one week later by AMPK- or EV-CARTs. Leukemia burden, via IVIS imaging, and survival were followed over time. Results: CD4+ CARTs co-transduced with AMPKγ2 (AMPK-CARTs) increased oxygen consumption, maximal respiration, and spare respiratory capacity following overnight stimulation by ~20% compared to CARTs co-transduced with the EV (EV-CARTs) (p&amp;lt;0.05). Following expansion, CD62L+CCR7+ central memory cells were 4.5-fold more abundant in the AMPK-CART group (2.4-6.2-fold, p&amp;lt;0.01). In vitro, while AMPK-CARTs demonstrated similar cytotoxicity against NALM6 targets as EV-CARTs in standard media, they mediated faster killing of NALM6 targets when challenged in tumor conditioned media (p&amp;lt;0.001). In vivo, recipients of AMPK-CARTs showed a consistent decrease in tumor burden as measured by median radiance values, translating to a significant survival advantage (p&amp;lt;0.05). Conclusions: Increasing AMPK signaling in CD4+ CARTs via overexpression of AMPKγ2 improved metabolic profile, increased memory generation, and enhanced toxicity against leukemia targets both in vitro and in vivo. Together, these features make elevating AMPK signaling an attractive method to increase the efficacy of CART therapy. Citation Format: Erica Braverman, Herbert Schuler, Mengtao Qin, Craig Byersdorfer. Driving AMPK signaling in CD4+ CART cells enhances metabolic capacity, memory formation, and anti-leukemia activity [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 45.

Abstract 5370: Tumor-intrinsic cGAS-STING activation promotes anti-tumor inflammatory response in osteosarcoma

Abstract A “cold” tumor microenvironment (TME) is often seen in patients with osteosarcoma (OS). Genomic instability, characteristic of OS, can stimulate the cGAS-STING pathway and activate the innate immune system. Tumor-intrinsic STING pathway silencing mechanisms can link genomic instability to immune evasion in OS. We hypothesize that dysregulation of the cGAS-STING pathway suppresses an innate immune response to genomic instability and plays a key role in tumor progression through establishing an immunosuppressive TME in OS. Using a novel panel of 11 OS patient-derived cell lines, we have identified two groups in terms of capacity for tumor-intrinsic STING activation, and we aim to unravel the mechanism of STING repression in a subset of OS tumors. To investigate the effects of tumor-intrinsic STING activation on macrophage polarization we employed tumor conditioned media (TCM) assays with murine-derived macrophages and tumor lines treated with cGAS-STING agonism. Tumor-intrinsic STING activation resulted in a significant increase in pro-inflammatory macrophages in a manner dependent on tumor STING. Lastly, we performed bulk RNA seq of OS PDX cell lines treated with STING agonist, defining for the first time the OS-specific STING activation signature. We then evaluated primary patient samples for evidence of this signature by single sample GSEA and performed survival analysis after stratifying samples as high or low for this signature, which demonstrated a significant protective effect of the STING activation signature on progression-free and overall survival. We have defined two subtypes of OS in terms of responsiveness to STING activation and demonstrated that STING activation has a protective effect in the human disease, which is the foundation of current efforts to identify targetable mechanism to reverse STING unresponsiveness and activate immune surveillance in this cancer. Citation Format: Elizabeth "Betsy" Peale Young, Christine Johnson, Alex G. Lee, Eric Alejandro Sweet-Cordero. Tumor-intrinsic cGAS-STING activation promotes anti-tumor inflammatory response in osteosarcoma [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 5370.

ALDH3A1 upregulation inhibits neutrophils N2 polarization and halts oral cancer growth.

Tumor-associated neutrophils (TANs) are among the most abundant inflammatory cells in tumor microenvironment (TME). Aldehyde dehydrogenase 3A1 (ALDH3A1) is significantly reduced in oral squamous cell carcinoma (OSCC), ALDH3A1 overexpression suppresses tumorigenesis by inhibiting inflammation. This study investigated the relationship and mechanisms underlying the crosstalk between ALDH3A1 and TANs in OSCC. Immunohistochemistry and immunofluorescence were performed to investigate the abundance of TANs and the expression of ALDH3A1. dHL-60 were induced with tumor-conditioned media and recombinant IL-6/IL-8. The expression of key proteins in PI3K/AKT/NF-κB pathway were detected by RT-PCR and western blot. A xenograft model was utilized to examine the effect of ALDH3A1 on tumorigenicity and polarization of TANs. In patients with OSCC, TANs significantly increased and were associated with a worse prognosis. Additionally, ALDH3A1 negatively correlated with TANs infiltration and especially the N2 phenotype which was the prominent part in OSCC. Furthermore, our study demonstrated that tumor-derived IL-8 drives ALDH3A1-mediated TANs N2 polarization in the TME through PI3K/AKT/NF-κB pathway invitro and invivo. Our results indicate that TANs can serve as a prognostic biomarker and ALDH3A1 could be a promising therapeutic target for regulating TANs N2 polarization in antitumor therapy.

Abstract B036: cGAS-STING activation promotes anti-tumor inflammatory response in osteosarcoma: Implications for tumor microenvironment reprogramming and tumor progression in patients

Abstract A “cold” tumor microenvironment (TME) is often seen in pediatric patients with osteosarcoma (OS). Genomic instability, characteristic of OS, can stimulate the cGAS-STING pathway and activate the innate immune system. However, tumor-intrinsic STING pathway silencing mechanisms link genomic instability to immune evasion in OS. We hypothesized that dysregulation of the cGAS-STING pathway suppresses an innate immune response to genomic instability and plays a key role in tumor progression by shaping an immunosuppressive TME in pediatric OS patients. Using a panel of 11 OS patient-derived xenograft (PDX)-derived cell lines, we characterized cGAS-STING pathway function and observed heterogeneity in STING responsiveness. Comprehensive biochemical evaluation of the inflammatory signaling response to STING agonism revealed two groups (6 responsive and 5 unresponsive) among our panel of 11 OS PDX-derived cell lines. To investigate the effects of OS tumor-intrinsic STING activation on macrophage polarization, we used tumor conditioned media (TCM) assays. We hypothesized that cGAS-STING activation in OS cell lines would be sufficient to shift macrophage phenotype ratios, enriching for a higher proportion of inflammatory macrophages, through the secretion of inflammatory factors that stimulate innate immune cells. A cGAS agonist, G3-YSD, was used to stimulate the STING pathway in murine OS lines. Addition of the TCM to macrophages resulted in a decrease in CD206+ “M2-like” macrophages and an increase in MHC II+ “M1-like” macrophages, measured via flow cytometry, in a manner dependent on tumor STING. Lastly, we performed bulk RNA sequencing of OS PDX cell lines treated with a STING agonist, defining for the first time the OS-specific STING activation signature. We then evaluated primary OS patient samples for evidence of this signature by gene set enrichment analysis and performed Kaplan-Meier survival analysis after stratifying samples as high or low for this signature, which demonstrated a significant protective effect of the STING activation signature on progression-free and overall survival. Together, we have shown that dysregulation of the cGAS-STING pathway represses the innate immune response to the genomic instability present in many OS cells and represents a key factor in the establishment of aggressive behavior in OS tumors. We have defined two subtypes of OS in terms of responsiveness to STING activation and demonstrated that STING activation has a protective effect in the human disease, which is the foundation of current efforts to explore targetable mechanisms to reverse STING unresponsiveness and activate immune surveillance in this cancer. Citation Format: Elizabeth P Young, Christine A Johnson, Alex G Lee, Courtney R Schott, E. Alejandro Sweet-Cordero. cGAS-STING activation promotes anti-tumor inflammatory response in osteosarcoma: Implications for tumor microenvironment reprogramming and tumor progression in patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B036.

Gab2 promotes the growth of colorectal cancer by regulating the M2 polarization of tumor‑associated macrophages.

Tumor‑associated macrophages (TAMs) are pivotal components in colorectal cancer (CRC) progression, markedly influencing the tumor microenvironment through their polarization into the pro‑inflammatory M1 or pro‑tumorigenic M2 phenotypes. Recent studies have highlighted that the Grb2‑associated binder 2 (Gab2) is a critical gene involved in the development of various types of tumor, including CRC. However, the precise role of Gab2 in mediating TAM polarization remains incompletely elucidated. In the present study, it was discovered that Gab2 was highly expressed within CRC tissue TAMs, and was associated with a poor prognosis of patients with CRC. Functionally, it was identified that the tumor‑conditioned medium (TCM) induced Gab2 expression, facilitating the TAMs towards an M2‑like phenotype polarization. Of note, the suppression of Gab2 expression using shRNA markedly inhibited the TCM‑induced expression of M2‑associated molecules, without affecting M1‑type markers. Furthermore, the xenotransplantation model demonstrated that Gab2 deficiency in TAMs inhibited tumor growth in the mouse model of CRC. Mechanistically, Gab2 induced the M2 polarization of TAMs by regulatingthe AKT and ERK signaling pathways, promoting CRC growth and metastasis. In summary, the present study study elucidates that decreasing Gab2 expression hinders the transition of TAMs towards the M2 phenotype, thereby suppressing the growth of CRC. The exploration of the regulatory mechanisms of Gab2 in TAM polarization may enhance the current understanding of the core molecular pathways of CRC development and may thus provide a foundation for the development of novel immunotherapeutic strategies targeted against TAMs.

Bone-homing metastatic breast cancer cells impair osteocytes’ mechanoresponse in a 3D loading model

Breast cancer predominantly metastasizes to the skeleton. Mechanical loading is reliably anabolic in bone, and also inhibits bone metastatic tumor formation and bone loss in vivo. To study the underlying mechanisms, we developed a 3D culture model for osteocytes, the primary bone mechanosensor. We verified that MLO-Y4s responded to perfusion by reducing their rankl and rankl:opg gene expression. We next cultured MLO-Y4s with tumor-conditioned media (TCM) collected from human breast cancer cells (MDA-MB-231s) and a corresponding bone-homing subclone to test the impacts on osteocytes’ mechanosensation. We found that TCM from the bone-homing subclone was more detrimental to MLO-Y4 growth and viability, and it abrogated loading-induced changes to rankl:opg. Our studies demonstrate that MLO-Y4s, including their mechanoresponse to perfusion, were more negatively impacted by soluble factors from bone-homing breast cancer cells compared to those from parental cells.

TGF-β1 activates neutrophil signaling and gene expression but not migration.

Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. The presence of transforming growth factor-beta (TGF-β) in the tumor microenvironment reportedly contributes to the skewing of neutrophils to a more pro-tumor phenotype. The effects of TGF-β on neutrophil signaling and migration are, however, unclear. We sought to characterize TGF-β signaling in both primary human neutrophils and the neutrophil-like cell line HL-60 and determine whether it directly induces neutrophil migration. We found that TGF-β1 does not induce neutrophil chemotaxis in transwell or underagarose migration assays. TGF-β1 does activate canonical signaling through SMAD3 and noncanonical signaling through ERK1/2 in neutrophils in a time- and dose-dependent manner. Additionally, TGF-β1 present in the tumor-conditioned media (TCM) of invasive breast cancer cells results in SMAD3 activation. We discovered that TCM induces neutrophils to secrete leukotriene B4 (LTB4), which is a lipid mediator important for amplifying the range of neutrophil recruitment. However, TGF-β1 alone does not induce secretion of LTB4. RNA-sequencing revealed that TGF-β1 and TCM alter gene expression in HL-60 cells, including the mRNA levels of the pro-tumor oncostatin M (OSM) and vascular endothelial growth factor A (VEGFA). These new insights into the role and impact of TGF-β1 on neutrophil signaling, migration, and gene expression have significant implications in the understanding of the changes in neutrophils that occur in the tumor microenvironment.

Siglec-9+ tumor-associated macrophages delineate an immunosuppressive subset with therapeutic vulnerability in patients with high-grade serous ovarian cancer

BackgroundThe potent immunosuppressive properties of sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) on myeloid cells and lymphocytes provide a strong rationale for serving as a therapeutic target. However, the expression profile and...

SONIC HEDGEHOG (SHH) PROMOTES BLOOD-BRAIN BARRIER (BBB) INTEGRITY IN DIFFUSE MIDLINE GLIOMA OF THE PONS

Abstract Diffuse Midline Glioma of the Pons (DMG-P) is the most common brainstem tumor in children and is universally lethal in this age group. The intact Blood-Brain Barrier (BBB) in these tumors are believed to play a major role in preventing systemically administered therapeutic agents from reaching therapeutic concentrations in the tumor. The factors that contribute to the integrity of BBB in DMG-P is not well understood. Sonic Hedgehog (SHH) signaling promotes BBB integrity in the developing and adult brain and plays an important role in the formation and maintenance of DMG-P tumors. In the present study, we show that DMG-P tumor cells secrete SHH ligand. Secreted SHH present in the tumor conditioned media (TCM) tightens the BBB by increasing electrical impedance and decreased paracellular and transcellular permeability in BBB Endothelial cells (ECs). Mechanistically, SHH in TCM causes tightening of the BBB by increasing the expression of junctional proteins in BBB-ECs, likely by signaling through non- canonical pathways. SHH in the TCM also increased the expression of ATP-binding cassette (ABC) transporter proteins ABCB1(P- glycoprotein) and ABCG2 (BCRP- Breast cancer resistance protein) in the Endothelial cells. These pro-barrier functions of SHH in TCM can be reversed by pharmacological inhibition of the S-acylation of the SHH ligand using Hedgehog acyltransferase (HHAT) inhibitor RUSKI-201. Our study demonstrates a potential therapeutic strategy of altering the BBB permeability to increase the efficacy of systemic therapies in DMG-P tumors. Ongoing studies will validate these findings in orthotopic murine models of DMG-P.

THE ROLE OF ICAM1 IN TUMOR ASSOCIATED MACROPHAGES IN GLIOBLASTOMA TUMORIGENESIS

Abstract Glioblastoma (GBM) is a fatal brain cancer in adults with ineffective treatment methods. Cell adhesion molecules (CAMs) are proteins that are expressed on the surface of cells and enable them to interact with one another and the surrounding microenvironment. Intracellular adhesion molecule 1 (ICAM-1) is a cell adhesion molecule expressed by various cell types. Preliminary data showed that ICAM-1 is associated with poorer overall and progression free survival in patients and ICAM-1 expression levels increase in recurrent tumors. TAMs enhance GBM tumor growth. The aim of this study was to determine if ICAM-1 expression on TAMs contributes to GBM tumorigenesis, specifically within the hypoxic tumor microenvironment. We hypothesized that ICAM1 facilitates TAM migration, proliferation, cell adhesion and phagocytosis, thus enhancing tumor growth. We found that upon incubation of human and mouse primary macrophages in hypoxia, ICAM-1 expression levels increased and were further exasperated upon culturing with tumor cell-conditioned medium. The migration, cell adhesion and phagocytosis of ICAM-1 deficient macrophages was lower than wild type macrophages, and effects were further exasperated upon culturing with tumor conditioned media and incubation in hypoxia. Intracranial injection of tumor cells into ICAM-1 deficient and wild type mice revealed that ICAM1 deficient mice survived longer with lower overall tumor volume than wild type mice, with survival and volume differences being rescued upon ICAM1 bone marrow reconstitution. In conclusion, the expression of ICAM1 in TAMs likely promotes GBM tumorigenesis and hypoxia further enhances the expression of ICAM-1 in macrophages which is associated with more aggressive GBM.