Microenvironmental Factors Research Articles

Endometriosis-Associated Ovarian Cancer: From Molecular Pathologies to Clinical Relevance.

Endometriosis is a chronic condition affecting reproductive-aged women, characterized by the growth of ectopic endometrial tissue. Despite being benign, endometriosis is associated with an increased risk of certain cancers, including endometriosis-associated ovarian cancer (EAOC). Ovarian cancer is rare, but more common in women with endometriosis, particularly endometrioid and clear-cell carcinomas. Factors such as hormonal imbalance, reproductive history, environmental exposures, and genetic predisposition contribute to the malignant transformation of endometriosis. Thus, understanding potential risk factors causing malignancy is crucial. Over the past few decades, various genetic mutations, microRNAs, as well as tumor microenvironmental factors have been identified, impacting pathways like PI3K/AKT/mTOR, DNA repair mechanisms, oxidative stress, and inflammation. Thus, this review aims to summarize molecular studies involved in EAOC pathogenesis as potential therapeutic targets. However, further research is needed to better understand the molecular and environmental factors driving EAOC development, to target the susceptibility of endometriotic lesions to malignant progression, and to identify effective therapeutic strategies.

Macrophage-coated tumor cluster aggravates hepatoma invasion and immunotherapy resistance via generating local immune deprivation

Immune checkpoint inhibitors (ICIs) represent a promising treatment for hepatocellular carcinoma (HCC) due to their capacity for abundant lymphocyte infiltration. However, some patients with HCC respond poorly toICI therapy due to the presence of various immunosuppressive factors in the tumor microenvironment. Our research reveals that a macrophage-coated tumor cluster (MCTC) signifies a unique spatial structural organization in HCC correlating with diminished recurrence-free survival and overall survival in a total of 572 HCC cases from 3 internal cohorts and 2 independent external validation cohorts. Mechanistically, tumor-derived macrophage-associated lectin Mac-2 binding protein (M2BP) induces MCTC formation andtraps immunocompetent cells at the edge of MCTCs to induce intratumoral cytotoxic Tcell exclusion and local immune deprivation. Blocking M2BP with a Mac-2 antagonist might provide an effective approach to prevent MCTC formation, enhance Tcell infiltration, and thereby improve the efficacy of ICI therapy in HCC.

The role of transcription factor FOXA1/C2/M1/O3/P1/Q1 in breast cancer.

Breast cancer is a common malignancy with the highest mortality rate among women worldwide. Its incidence is on the rise year after year, accounting for more than one-tenth of new cancers worldwide. Increasing evidence suggests that forkhead box (FOX) transcription factors play an important role in the occurrence and development of breast cancer. However, little is known about the relationship between the expression, prognostic value, function, and immune infiltration of FOX transcription factors in tumor microenvironment. We used bioinformatics to investigate expression and function of FOX factor in breast cancer. Our results revealed the expression levels of FOXA1 and FOXM1 were significantly higher in breast cancer tissues than in normal tissues. The high expression of mRNA in FOXA1 (P < .05), FOXM1 (P < .01), and FOXP1 (P < .05) groups was related to tumor stage. Survival analysis results showed that increased FOXP1 mRNA levels were significantly associated with overall survival (OS), recurrence-free survival (RFS), and distant metastasis-free survival (DMFS) in all patients with breast cancer (P < .05). Patients with the FOXA1 high-expression group had better RFS and DMFS than the low-expression group (P < .05), while patients with FOXM1 high-expression group had worse RFS, OS, and DMFS than the low-expression group (P < .05). Meanwhile, mutation analysis showed that genetic alterations in FOX transcription factors were significantly associated with shorter OS and progression-free survival (P < .05), but not with disease-free survival (P = .710) in patients with breast cancer. FOXP1, FOXA1, and FOXM1 may be used as potential biomarkers to predict the prognosis of patients with breast cancer. Functional enrichment indicated that FOX was mainly involved in cell division, cell senescence, cell cycle, and prolactin signaling pathway. In patients with breast cancer, FOXC2 expression was negatively correlated with the infiltration of B cells and positively correlated with the infiltration of neutrophils and dendritic cells. However, FOXM1 was negatively correlated with the infiltration of CD8 + T cells and macrophages and positively correlated with the infiltration of neutrophils and dendritic cells. These findings provided novel insights into the screening of prognostic biomarkers of the FOX family in breast cancer and laid a foundation for further research on the immune infiltration of the FOX transcription factor family members in tumors.

Vitamin A-treated natural killer cells reduce interferon-gamma production and support regulatory T-cell differentiation.

Natural killer (NK) cells are innate cytotoxic lymphocytes that contribute to immune responses against stressed, transformed, or infected cells. NK cell effector functions are regulated by microenvironmental factors, including cytokines, metabolites, and nutrients. Vitamin A is an essential micronutrient that plays an indispensable role in embryogenesis and development, but was also reported to regulate immune responses. However, the role of vitamin A in regulating NK cell functions remains poorly understood. Here, we show that the most prevalent vitamin A metabolite, all-trans retinoic acid (atRA), induces transcriptional and functional changes in NK cells leading to altered metabolism and reduced IFN-γ production in response to a wide range of stimuli. atRA-exposed NK cells display a reduced ability to support dendritic cell (DC) maturation and to eliminate immature DCs. Moreover, they support the polarization and proliferation of regulatory T cells. These results imply that in vitamin A-enriched environments, NK cells can acquire functions that might promote tolerogenic immunity and/or immunosuppression.

Advances in molecular mechanisms of inflammatory bowel disease‑associated colorectal cancer (Review).

The link between inflammation and cancer is well documented and colonic inflammation caused by inflammatory bowel disease (IBD) is thought to be a high-risk factor for the development of colorectal cancer (CRC). The complex crosstalk between epithelial and inflammatory cells is thought to underlie the progression from inflammation to cancer. The present review collates and summarises recent advances in the understanding of the pathogenesis of IBD-associated CRC (IBD-CRC), including the oncogenic mechanisms of the main inflammatory signalling pathways and genetic alterations induced by oxidative stress during colonic inflammation, and discusses the crosstalk between the tumour microenvironment, intestinal flora and host immune factors during inflammatory oncogenesis in colitis-associated CRC. In addition, the therapeutic implications of anti-inflammatory therapy for IBD-CRC were discussed, intending to provide new insight into improve clinical practice.

The shaping of mRNA translation plasticity by RNA G-quadruplexes in cancer progression and therapy resistance.

Translational reprogramming in response to oncogenic signaling or microenvironmental stress factors shapes the proteome of cancer cells, enabling adaptation and phenotypic changes underlying cell plasticity, tumor progression and response to cancer therapy. Among the mechanisms regulating translation are RNA G-quadruplexes (RG4s), non-canonical four-stranded structures whose conformational modulation by small molecule ligands and RNA-binding proteins affects the expression of cancer proteins. Here, we discuss the role of RG4s in the regulation of mRNA translation by focusing on paradigmatic examples showing their contribution to adaptive mechanisms of mRNA translation in cancer.

Abstract CT287: LINNOVATE: A Phase 1/2 study of safety/efficacy using lurbinectedin, combined with ipilimumab, and nivolumab for advanced soft tissue sarcomas (NCT05876715)

Abstract Background: Sarcoma cells are most immunogenic earlier in the course of the disease. Therefore, immune checkpoint inhibitors such as ipilimumab (ipi) and nivolumab (nivo), that promote sustained T-cell activation by suppressing T regs would be most effective when given as first line therapy, together with a tumoricidal agent, such as lurbinectedin, whose plausible mechanism of action is not only to destroy the cancer cells and expose the tumor neoantigens for immune recognition, but also to destroy growth promoting factors in the tumor microenvironment. In the SAINT phase 2 study using ipi, nivo and trabectedin as first line therapy for advanced soft tissue sarcomas (STS; n=79), there were 6 complete responses, 14 partial responses, 49 stable disease, 25.3% best response rate, 87.3% disease control rate; median PFS, 6.7 months, median OS, 24.6 months (Gordon et al, Cancers vol. 15, 906, 2023). Lurbinectedin is a synthetic marine-derived agent and an analog of trabectedin. Therefore, we expect that lurbinectedin will be as effective as trabectedin when used together with ipi and nivo as first line therapy for advanced STS. Methods: Objective 1: Assess MTD of lurbinectedin. Objective 2: Evaluate PFS, OS, incidence and severity of adverse events; Correlative: Correlate response with ctDNA using Signatera. This is an open label, dose-seeking phase 1/2 study. The phase 1 part employs standard “cohort of three” design with a DLT window of 6 weeks. Treatment schedule: Ipi 1 mg/kg IV q 12 weeks; Nivo 3 mg/kg IV q 2 weeks; escalating doses of lurbinectedin from 2.6 mg/m2 to 3.2 mg/m2 IV q 3 weeks. Phase 2 part follows completion of phase 1 part wherein additional 28-34 participants will receive lurbinectedin at the MTD with fixed doses of Ipi and Nivo. Participants may continue treatment until significant disease progression or unacceptable toxicity occurs. Eligible patients are 18 years of age or older, previously treated in phase 1 and previously untreated in phase 2 with confirmed diagnosis of advanced STS, adequate hematologic and organ function, and no history of autoimmune disorder. To date, 6 patients in phase 1 have been dosed. Citation Format: Erlinda M. Gordon, Nadezhda Omelchenko, Samantha Jeffrey, Vaishali Kumar, Neal S. Chawla, Mahesh Seetharam, Victoria Chua, Ania Moradkhani, Doris Quon, Steven Wong, Sant Chawla. LINNOVATE: A Phase 1/2 study of safety/efficacy using lurbinectedin, combined with ipilimumab, and nivolumab for advanced soft tissue sarcomas (NCT05876715) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT287.

Abstract LB121: Endothelial cells mediated paracrine signaling alters immune cell modulators on colorectal cancer cells

Abstract Background: Metastatic colorectal cancer (mCRC) is the second leading cause of cancer related deaths in the US. Various factors in the tumor microenvironment (TME) play significant roles in progression of CRC tumors and their response to various therapies. Previous studies from our laboratory and other groups have demonstrated that blood vessels and their constituent endothelial cells (ECs) exert strong effects on CRC cells by altering stemness, enhancing drug resistance, and increasing cell survival. Although ECs have also been shown to affect immune cell function or alter the tumor immune environment by direct or indirect mechanisms, their role in altering immune-modulating effectors on tumor cells, especially CRC cells, are poorly understood. Thus, elucidating the role of ECs in regulating immune-modulators in CRC cells and understanding the underlying mechanisms by which this occurs will provide important insights into the role of ECs on altering the immune-microenvironment of CRC tumors that likely affects tumor progression and, possibly, response to immunotherapies. Methods: A panel of CRC cells lines were exposed to conditioned media (CM) from CRC or multiple primary or immortalized liver parenchymal endothelial cells (LPECs), and alterations in some of the known immune-inhibitory ligands were measured by western blotting or flow cytometry. Molecular size-based fractionation of CRC-CM or LPEC-CM were performed and various sized fractions were utilized to detect fractions harboring inducers of immune-inhibitory ligands. Mass spectrometry was performed to identify possible factors in LPEC-CM. Western blotting of CRC cell lysates was performed to identify possible signaling changes following LPEC-CM treatment. Results: Our studies demonstrated that ECs were more efficient in inducing PD-L1 expression in CRC cells compared to other cell types present in the CRC tumor microenvironment. LPEC-CM induced PD-L1 expression in multiple CRC cells regardless of their genetic mutations or MSI-H/MSS status. Initial studies indicate that LPEC-CM can also enhance MHC-1 expression in CRC cells. Size-fractionation studies indicate that inducer/s of PD-L1 expression are present in a fraction &amp;gt;100kDa. Mass-spectrometry of this fraction has identified prospective candidates that can possibly induce PD-L1 on CRC cells. CRC cells treated with LPEC-CM led to enhanced levels of pSTAT3, a known PD-L1 transcription factor. Conclusions: Our studies, for the first time, demonstrate that ECs, through paracrine signaling, can educate CRC cells to overexpress immune-modulatory ligands. Further studies are being conducted to elucidate the molecular factors involved in these processes. These studies not only shed light on the complex role played by ECs in modulating the TME, but also warrants further investigations into the effects of these changes in CRC tumor progression and responses to therapies. Citation Format: FanFan, Lee M. Ellis, Rajat Bhattacharya. Endothelial cells mediated paracrine signaling alters immune cell modulators on colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB121.

Conserved methylation signatures associate with the tumor immune microenvironment and immunotherapy response

BackgroundAberrant DNA methylation is a major characteristic of cancer genomes. It remains unclear which biological processes determine epigenetic reprogramming and how these processes influence the variants in the cancer methylome, which can further impact cancer phenotypes.MethodsWe performed pairwise permutations of 381,900 loci in 569 paired DNA methylation profiles of cancer tissue and matched normal tissue from The Cancer Genome Atlas (TCGA) and defined conserved differentially methylated positions (DMPs) based on the resulting null distribution. Then, we derived independent methylation signatures from 2,465 cancer-only methylation profiles from the TCGA and 241 cell line-based methylation profiles from the Genomics of Drug Sensitivity in Cancer (GDSC) cohort using nonnegative matrix factorization (NMF). We correlated DNA methylation signatures with various clinical and biological features, including age, survival, cancer stage, tumor immune microenvironment factors, and immunotherapy response. We inferred the determinant genes of these methylation signatures by integrating genomic and transcriptomic data and evaluated the impact of these signatures on cancer phenotypes in independent bulk and single-cell RNA/methylome cohorts.ResultsWe identified 7,364 differentially methylated positions (2,969 Hyper-DMPs and 4,395 Hypo-DMPs) in nine cancer types from the TCGA. We subsequently retrieved three highly conserved, independent methylation signatures (Hyper-MS1, Hypo-MS1, and Hypo-MS4) from cancer tissues and cell lines based on these Hyper and Hypo-DMPs. Our data suggested that Hypo-MS4 activity predicts poor survival and is associated with immunotherapy response and distant tumor metastasis, and Hypo-MS4 activity is related to TP53 mutation and FOXA1 binding specificity. In addition, we demonstrated a correlation between the activities of Hypo-MS4 in cancer cells and the fractions of regulatory CD4 + T cells with the expression levels of immunological genes in the tumor immune microenvironment.ConclusionsOur findings demonstrated that the methylation signatures of distinct biological processes are associated with immune activity in the cancer microenvironment and predict immunotherapy response.

Integrated analyses for identification of a three-gene signature associated with Chaihu Shugan San formula for hepatocellular carcinoma treatment.

Chaihu Shugan San (CSS) is a well-known traditional herbal formula that has the potential to ameliorate hepatocellular carcinoma (HCC); however, its mechanism of action remains unknown. Here, we identified the key targets of CSS against HCC and developed a prognostic model to predict the survival of patients with HCC. The effect of CSS plus sorafenib on HCC cell proliferation was evaluated using the MTT assay. LASSO-Cox regression was used to establish a three-gene signature model targeting CSS. Correlations between immune cells, immune checkpoints and risk score were determined to evaluate the immune-related effects of CSS. The interactions between the components and targets were validated using molecular docking and Surface Plasmon Resonance (SPR) assays. CSS and sorafenib synergistically inhibited HCC cell proliferation. Ten core compounds and 224 targets were identified using a drug compound-target network. The prognostic model of the three CSS targets (AKT1, MAPK3 and CASP3) showed predictive ability. Risk scores positively correlated with cancer-promoting immune cells and high expression of immune checkpoint proteins. Molecular docking and SPR analyses confirmed the strong binding affinities of the active components and the target genes. Western blot analysis confirmed the synergistic effect of CSS and sorafenib in inhibiting the expression of these three targets. In conclusion, CSS may regulate the activity of immune-related factors in the tumour microenvironment, reverse immune escape, enhance immune responses through AKT1, MAPK3, and CASP3, and synergistically alleviate HCC. The co-administration of sorafenib with CSS has a strong clinical outlook against HCC.

Tumor Microenvironment in Colorectal Cancer Development: A Review of 3D Study Analysis

Background: Colorectal cancer is the third most common cancer in 2020, with a high mortality rate. Colorectal cancer treatment has made many advances. However, many factors in the tumor microenvironment still have not been reached but significantly affect the success of treatment. This literature review was conducted to search for research articles that analyze factors in the tumor microenvironment, how they interact through studies primarily conducted in 3D, and how to prepare for 3D research on colorectal cancer will be briefly discussed. The aim of this systematic review is to study the components of the tumor microenvironment in colorectal cancer development as assessed by 3D studies. Methods: Article searches were conducted through Embase, Scopus and PubMed. From the 110 articles found at the beginning of the search, after going through several screening stages, 27 articles were determined that met the inclusion criteria. The inclusion criteria used were journals containing research articles on colorectal cancer in the last five years with topics regarding the tumor microenvironment and according to keywords. Results: Microenvironment components in colorectal cancer, consisting of cellular and noncellular components, have the most significant effect on cancer development and ultimately affect metastasis, response to treatment, and prognosis. Conclusions: This literature review proves that the components of the tumor microenvironment are very diverse, making colorectal cancer heterogeneous. It still requires a lot of research to prove the existence of other components that affect the effectiveness of the treatment.

Molecular diversity in isocitrate dehydrogenase-wild-type glioblastoma.

In the dynamic landscape of glioblastoma, the 2021 World Health Organization Classification of Central Nervous System tumours endeavoured to establish biological homogeneity, yet isocitrate dehydrogenase-wild-type (IDH-wt) glioblastoma persists as a tapestry of clinical and molecular diversity. Intertumoural heterogeneity in IDH-wt glioblastoma presents a formidable challenge in treatment strategies. Recent strides in genetics and molecular biology have enhanced diagnostic precision, revealing distinct subtypes and invasive patterns that influence survival in patients with IDH-wt glioblastoma. Genetic and molecular biomarkers, such as the overexpression of neurofibromin 1, phosphatase and tensin homolog and/or cyclin-dependent kinase inhibitor 2A, along with specific immune cell abundance and neurotransmitters, correlate with favourable outcomes. Conversely, increased expression of epidermal growth factor receptor tyrosine kinase, platelet-derived growth factor receptor alpha and/or vascular endothelial growth factor receptor, coupled with the prevalence of glioma stem cells, tumour-associated myeloid cells, regulatory T cells and exhausted effector cells, signifies an unfavourable prognosis. The methylation status of O6-methylguanine-DNA methyltransferase and the influence of microenvironmental factors and neurotransmitters further shape treatment responses. Understanding intertumoural heterogeneity is complemented by insights into intratumoural dynamics and cellular interactions within the tumour microenvironment. Glioma stem cells and immune cell composition significantly impact progression and outcomes, emphasizing the need for personalized therapies targeting pro-tumoural signalling pathways and resistance mechanisms. A successful glioblastoma management demands biomarker identification, combination therapies and a nuanced approach considering intratumoural variability. These advancements herald a transformative era in glioblastoma comprehension and treatment.

Unveiling the immunosuppressive landscape of pancreatic ductal adenocarcinoma: implications for innovative immunotherapy strategies.

Pancreatic cancer, particularly pancreatic ductal adenocarcinoma (PDAC), stands as the fourth leading cause of cancer-related deaths in the United States, marked by challenging treatment and dismal prognoses. As immunotherapy emerges asapromising avenue for mitigating PDAC's malignant progression, a comprehensiveunderstanding of the tumor's immunosuppressive characteristics becomesimperative. This paper systematically delves into the intricate immunosuppressive network within PDAC, spotlighting the significant crosstalk between immunosuppressive cells and factors in the hypoxic acidic pancreatic tumor microenvironment. By elucidating these mechanisms, we aim to provide insights into potential immunotherapy strategies and treatment targets, laying the groundwork for future studies on PDAC immunosuppression. Recognizing the profound impact of immunosuppression on PDAC invasion and metastasis, this discussion aims to catalyze the development of more effective and targeted immunotherapies for PDAC patients.

Abstract 6944: Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer

Abstract Intratumor heterogeneity (ITH) is a substrate for tumor evolution and confers amenability to stressors and therapies. Sources of ITH include subclonal mutations, microenvironmental factors, and plasticity whereby a cell has the ability to adopt different cell states. Clear cell kidney cancer (ccRCC) has a well-characterized genetic initiating event - VHL inactivation - resulting in activation of HIF-1 and HIF-2 transcription factors. Although VHL inactivation is consistently clonal in nature, ccRCC nonetheless exhibits extensive genetic and morphological ITH. This makes ccRCC an excellent model in which to study ITH. We performed single cell RNA-seq on multiregional samples from kidney tumors and macroscopically normal kidney tissue. We obtained 55 samples from 16 tumors and used the 10x Genomics platform to obtain gene expression data from ~150,000 single cells, including ~80,000 cancer and normal epithelial cells. We identified three cancer cell states, co-existing in all clear cell kidney cancer tumors analyzed and marked by different gene expression programs: 1) differentiated-like cells marked by proximal tubule cell genes characteristic of the cancer cell of origin; 2) epithelial-mesenchymal transition (EMT)-like cells and 3) injured-like cells. Differentiated markers and EMT markers were associated with good and poor prognosis, respectively, indicating that these cell subpopulations are clinically important. Notably, these cell states are also present in the normal proximal tubule although in different proportions to the tumor samples, which contain more cells in the EMT-like and injured-like states. This suggests that the core features of ITH are inherent to the cell of origin of the cancer, but that the cell state trajectories are dysregulated in the tumor. Furthermore, in tumors these states exist as a continuum, whereby transitional cells in between states appear to exist, suggesting that cancer cells are continually and dynamically transitioning between states. Lastly, we used chromosomal copy number variation (CNV) patterns to study genetic ITH. We examined the effect of 14q loss, which is associated with ccRCC metastasis, on gene expression. We found that 14q loss is associated with downregulation of HIF-1 transcriptional activity together with a reciprocal increase in HIF-2 activity, MYC activity and oxidative phosphorylation, highlighting potential mechanisms by which 14q loss may driver ccRCC metastasis. Citation Format: Olivia Lombardi, Ran Li, Lisa Browning, Faiz Jabbar, Hannah Evans, Peter J. Ratcliffe, David R. Mole. Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer [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 6944.

Abstract 194: Single-cell mechanical analysis reveals that astrocytes and glioblastoma cells have similar viscoelastic properties

Abstract Despite recent advances in treatment, the survival rate of glioblastoma (GBM) patients remains low due to the cancer’s resistance to current treatment modalities. Solid tumors like GBM are known to harbor physical abnormalities at the tissue and cell level. An understanding of GBM cell mechanics may therefore lead to the identification of novel disease mechanisms, biomarkers, and therapeutic strategies to improve treatment outcomes. In this study, we aimed to compare the mechanics of human GBM cells and their normal counterpart, immortalized human astrocytes (IHAs). To accomplish this, we used a parallel-plate flow system to expose single cells to physiological fluid shear stress and recorded the resulting deformation using a camera-coupled microscope. We then utilized digital image correlation analysis to create strain-time profiles of individual cells and fit the profiles with a three-parameter generalized Maxwell model to characterize their nuclear and cytoplasmic viscoelastic properties. We also used fluorescence microscopy and atomic force microscopy to quantify actin density and alignment and adhesion energy in both cell types, respectively. Our results showed that, contrary to extracranial cancer cells, the viscoelastic properties of GBM cells are similar to their normal counterpart. This may explain why metastasis in GBM patients is rare and conventional treatment options are unsuccessful. We also found that actin organization and localization and adhesion energy is different between GBM cells and IHAs. Thus, future research which links the genetic and microenvironmental factors that modulate these unique mechanical features may lead to the development of rational and novel clinical strategies against GBM. Citation Format: Julian Najera, Killian Onwudiwe, Luke Holen, Alice A. Burchett, Dorielis Rodriguez, Maksym Zarodniuk, Saeed Siri, Meenal Datta. Single-cell mechanical analysis reveals that astrocytes and glioblastoma cells have similar viscoelastic properties [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 194.

Abstract 6837: Prolonged effects of hypoxia in luminal breast cancer cells

Abstract Metastasis is the main cause of tumor-related mortality in breast cancer patients. Improving our understanding of the metastatic cascade is imperative to improve patient outcomes. Prior research indicates cancer cells with increased metastatic potential have acquired adaptive properties induced by factors in the tumor microenvironment (TME), including hypoxia. However, previous hypoxia research has primarily focused on signaling changes that occur when cancer cells are in the hypoxic TME and does not account for long-term changes that may occur once cancer cells re-enter normoxia, such as when they metastasize. Our research in patient-derived circulating tumor cell (CTCs) lines and breast cancer cell lines indicates that certain transcriptional changes are maintained after cells leave hypoxia, and that these post-hypoxic cells maintain a higher metastatic potential compared to cells not exposed to hypoxia. Taking an un-biased approach to define changes in hypoxia-exposed breast cancer cells upon reoxygenation, we performed bulk RNA-Seq on post-hypoxic luminal breast cancer cell lines compared to normoxic controls cultured either in adherent or suspension conditions, the latter to mimic the physiological context of CTCs. We found that a subset of genes that were differentially expressed in hypoxic conditions were maintained as up- or down-regulated after cells were reoxygenated, indicating a “hypoxic memory”. Additionally, a subset of differentially expressed genes were unique to the post-hypoxic cells, demonstrating both prolonged and novel gene expression changes induced by hypoxia exposure. We also performed functional assays and observed that this post-hypoxic state is associated with a more aggressive phenotype. Current research in our lab is on-going to understand the epigenetic changes that may be responsible for this hypoxic memory. Overall, this research will improve our understanding of the prolonged consequences of hypoxia on CTC biology and metastasis. Citation Format: Aidan Moriarty, Oihana Iriondo, Yonatan Amzaleg, Remi Klotz, Min Yu. Prolonged effects of hypoxia in luminal breast cancer cells [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 6837.

Nanocomposite magnetic hydrogel with dual anisotropic properties induces osteogenesis through the NOTCH-dependent pathways

Physical factors in the cellular microenvironment have critical effects on stem cell differentiation. The utilization of physical factors to promote the osteogenic differentiation of stem cells has been established as a new strategy for developing bone tissue engineering scaffolds. In this context, scaffolds with multiscale anisotropy are considered to possess biomimetic properties, which are advantageous for their biological performance. In the present study, a novel magnetic anisotropic hydrogel (MAH) with magnetic and topographic anisotropy was designed by combining static magnetic field-induced magnetic nanomaterials and a hydrogel. In in vitro studies, the MAH exhibited excellent biocompatibility and osteogenic bioactivity. The alkaline phosphatase activity and the expression of osteogenic-related genes and proteins induced by the MAH were greater than those induced by the pure PEGDA–GelMA hydrogel (PGH) and the magnetic isotropic hydrogel (MIH). In addition, the present study revealed that the dual anisotropic properties of the MAH activated the NOTCH1/2 pathway by upregulating SNHG5 and downstream SIRT6, which modulates the level of NOTCH1/2 by antagonizing DNMT1 protein stability, ultimately inducing the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Furthermore, the MAH, MIH, and PGH were tested for in vivo bone regeneration in rabbits with femur defects, and the results demonstrated that the MAH effectively stimulated bone regeneration. Taken together, these findings suggest that this magnetically and topographically anisotropic biomimetic hydrogel might be a promising candidate for application in the field of bone tissue regeneration.

Abstract 5527: Spatiotemporal analysis of growth factor regulation of osteosarcoma AKT and ERK signaling dynamics in an ex vivo lung metastasis model

Abstract In the present study, we investigated the relationships between spatiotemporal distribution of growth factors in the lung microenvironment, metastatic osteosarcoma cell signaling, and intratumoral gene expression heterogeneity. Osteosarcoma is an aggressive bone malignancy that commonly metastasizes to the lungs, resulting in significant patient mortality. During metastasis, disseminated cancer cells are exposed to microenvironment-derived growth factors that result in signaling pathway activation. Two such pathways are MAPK/ERK and PI3K/AKT, which regulate complex transcriptional programs underlying cell proliferation and survival decisions. In this study, we utilized precision cut lung slices (PCLS) obtained from mice with established osteosarcoma metastases to ex vivo model associations between the spatiotemporal distributions of growth factors in the lung, osteosarcoma signaling dynamics, and intratumoral gene expression heterogeneity, with single cell precision. To accomplish this, we engineered osteosarcoma cells that co-expressed genetically-encoded ERK and AKT sensors, engrafted them into PCLS tissues, then recorded sensor dynamics via live-cell microscopy. These tissues were subsequently fixed and immuno-stained for measurement of growth factor expression in resident lung cells and spatial correlation with observed tumor cell ERK-AKT signaling dynamics. Using this approach, we found that growth factor expression was diffusely increased within areas bordering osteosarcoma metastases. FGF2 and IGF1, which we have previously demonstrated to potently induce ERK signaling in osteosarcoma cells, were both significantly upregulated in these regions. However, despite relatively uniform overexpression of growth factors adjacent to tumors, individual osteosarcoma cells displayed stochastics patterns of ERK and AKT signaling, and downstream gene targets. These findings revealed that upregulation of lung growth factors, and the inherent systems-level properties of osteosarcoma signaling networks, can converge to promote intratumoral signaling and gene expression heterogeneity. Together, our results, using a novel PCLS and live-cell biosensor imaging approach, provide new insight into how spatial and temporal changes in microenvironmental signaling composition are integrated by metastatic osteosarcoma cells to promote heterogeneity. Citation Format: Rawan Makkawi, Ryan D. Roberts, Jeremy Copperman, Alexander E. Davies. Spatiotemporal analysis of growth factor regulation of osteosarcoma AKT and ERK signaling dynamics in an ex vivo lung metastasis model [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 5527.

Abstract 5535: Intracellular negative regulators of RTK-Ras-ERK signaling alter breast cancer perception of metastatic niche-derived growth factors

Abstract Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype that commonly metastasizes to distant organs, such as the lung, resulting in the poor clinical outcomes. TNBC cells commonly overexpress epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK) which when bound by microenvironmental growth factors, results in the activation of downstream effector kinases ERK-AKT and initiates transcriptional programs that control cell fate. Tumor cells, however, simultaneously receive many growth factor inputs from their respective microenvironments, many of which stimulate RTKs other than EGFR, that converge at ERK and AKT resulting in different transcriptional and cell fate outputs. This raises questions as to how tumor cells perceive, decode, and respond to a complex set of microenvironmental signals and what the relevance of this process may be for disease pathogenesis and treatment. To this end, our study aimed to determine how disseminated breast cancer cells perceive and respond to a complex growth factor milieu upon seeding and engraftment in the lung. Using live-cell microscopy techniques and TNBC cells carrying biosensors for ERK and Akt signaling pathways, we monitored the dynamic signaling behaviors of single tumor cells, in real-time, as they adapted to the environment in living human lung tissue. Strikingly, we found that individual disseminated tumor cells are less responsive to growth factors once seeded in the lung, as compared to outside this tissue, despite the presence of RTK stimulating ligands. We showed that partial activation of EGFR is necessary for complete activation of ERK signaling by other growth factor-RTK pairs, and expression of downstream ERK target genes, whereas AKT signaling was unaffected. Using quantitative modeling and experimentation, we showed that reshaping of ERK signaling response dynamics occurred via dysregulation of negative regulators that are dominantly controlled by EGFR signaling. Together, these results provide novel insight into how tumor cells perceive and respond to complex microenvironmental signals, which has important implications for drug targeting strategies. Citation Format: Vaibhav Murthy, Cemal Erdem, Jeremy Copperman, Marc Birtwistle, Alexander Davies. Intracellular negative regulators of RTK-Ras-ERK signaling alter breast cancer perception of metastatic niche-derived growth factors [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 5535.

Abstract 6319: Characterization of AB-1015 logic-gated CAR induction (ON kinetics), receptor turnover (OFF kinetics), and logic gate sensitivity to ALPG/P and MSLN

Abstract CAR T cell activity in solid tumors is limited, in part, by off-tumor toxicity. To address this obstacle, AB-1015 contains an “AND” logic gate, consisting of a priming receptor (PrimeR) and an inducible chimeric antigen receptor (CAR) that is upregulated by PrimeR activation. The AB-1015 logic gate targets ALPG/P and MSLN, which are coexpressed in ovarian tumors but not normal tissues. AB-1015 CAR induction within the tumor is expected to be a dynamic process influenced by multiple factors, including antigen density, target cell abundance, as well as tumor microenvironmental factors. To characterize CAR induction and receptor turnover, we developed an in vitro assay with plate-bound ALPG to induce CAR. CAR induction occurred quickly in response to ALPG priming, with most ICT cells expressing CAR within 24 hrs, and plateaued by 72 hrs. Once removed from ALPG, CAR expression returned to baseline within 96 hrs. Similar to conventional CAR T cells, the presence of the cytolytic target antigen (MSLN) was expected to increase the rate of CAR turnover on AB-1015. To assess this, we measured the CAR functional half-life of AB-1015 cells once removed from ALPG priming. The cytotoxic activity of AB-1015 cells rapidly decreased in the absence of ALPG, reaching baseline within 24 hrs. Of note, our in vitro assay did not result in 100% CAR induction from all PrimeR+ AB-1015 cells. To understand whether this reflected biology versus a limitation of our in vitro assay, we conducted a CAR induction, sorted the CAR- cells, then performed a second induction. ICT cells that did not express CAR after the first induction were shown to be able to induce CAR during the second induction assay, thereby demonstrating that all PrimeR+ ICT cells have the potential to induce CAR. To assess the sensitivity of AB-1015 for low density ALPG/P and MSLN, isogenic cell lines were engineered to express each antigen at variable levels. In co-culture, AB-1015 was able to prime and kill targets expressing ALPG and MSLN at or below the limit of detection using immunohistochemistry. In light of these observations, the specificity of AB-1015 was verified using a dual flank in vivo model where one tumor expressed both ALPG and MSLN, and the contralateral tumor expressed MSLN alone. While robust anti-tumor activity was observed against the ALPG+/MSLN+ tumor, no activity was detected against the MSLN+ tumor. In summary, AB-1015 CAR induction and receptor turnover kinetics is a dynamic process. A reductionist in vitro model system was developed, enabling analysis of CAR induction and receptor turnover. Importantly, we demonstrated that all PrimeR+ ICT cells have the potential to induce CAR. Finally, the in vivo specificity of AB-1015 for ALPG+/MSLN+ tumors was established using a dual flank specificity model. Together, these data support the continued development of AB-1015 in a phase I clinical trial (NCT05617755). Citation Format: Xinyan Tang, Marvin Chew, Rakesh Sudhakar, Hongruo Yun, Doreen Sakamoto, Vibhavari Sail, Amanda Fearon, Jasper Williams, Jun Feng, Stephen Santoro. Characterization of AB-1015 logic-gated CAR induction (ON kinetics), receptor turnover (OFF kinetics), and logic gate sensitivity to ALPG/P and MSLN [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 6319.