Gene Expression In Tumor Cells Research Articles

Longitudinal molecular profiling elucidates immunometabolism dynamics in breast cancer

Although metabolic reprogramming within tumor cells and tumor microenvironment (TME) is well described in breast cancer, little is known about how the interplay of immune state and cancer metabolism evolves during treatment. Here, we characterize the immunometabolic profiles of tumor tissue samples longitudinally collected from individuals with breast cancer before, during and after neoadjuvant chemotherapy (NAC) using proteomics, genomics and histopathology. We show that the pre-, on-treatment and dynamic changes of the immune state, tumor metabolic proteins and tumor cell gene expression profiling-based metabolic phenotype are associated with treatment response. Single-cell/nucleus RNA sequencing revealed distinct tumor and immune cell states in metabolism between cold and hot tumors. Potential drivers of NAC based on above analyses were validated in vitro. In summary, the study shows that the interaction of tumor-intrinsic metabolic states and TME is associated with treatment outcome, supporting the concept of targeting tumor metabolism for immunoregulation.

Abstract RF01-02: A randomized Phase I pre-operative window trial of transdermal endoxifen in women planning mastectomy: evaluation of dermal safety, intra-mammary drug distribution, and biologic effects

Abstract Background: Breast cancer prevention requires only local exposure of the breast to active drug but oral preventive agents entail systemic exposure, with resultant adverse effects, limiting acceptance by high-risk women. Drug-delivery through the breast skin is an attractive option, but requires demonstration of dermal safety and drug distribution throughout the breast. We formulated the tamoxifen metabolite (E/Z)-endoxifen in a hydroalcoholic/oleic acid gel for transdermal delivery. We tested this in a placebo-controlled, double-blinded Phase I trial with dose escalation from 10 to 20 mg daily and evaluated dermal safety, intra-mammary drug distribution, and biologic effects in DCIS or invasive cancers. Methods: Women planning mastectomy for therapy for Stage 0-III breast cancer, or surgical risk reduction (N=32) were randomized (2:1) to placebo-gel or endoxifen-gel applied to the skin of both breasts for 3-5 weeks. Our primary endpoint, dermal safety was evaluated using the modified NCI Common Terminology Criteria for Adverse Events version 4. Endoxifen concentration, and modulation of circulating tamoxifen biomarkers were evaluated to demonstrate minimal systemic effects. Plasma and tissue concentration of endoxifen were determined using Liquid Chromatography – Tandem Mass Spectrometry. Tamoxifen-responsive circulating markers, insulin-like growth factor 1 (IGF1) and sex hormone-binding globulin (SHBG), were measured with enzyme-linked immunosorbent assays. Finally, we explored anti-cancer efficacy in tumors by measuring the modulation of tumor cell proliferation (Ki67 labeling index) and gene expression associated with tamoxifen response in proliferation (BIRC5, MKI67, MYBL2, CCNB1, AURKA), cell cycle (UBE2C, PLK1, CEP55, CDK1, RRM2, TOP2A, PTTG1), cancer-associated stromal genes of invasion (FN1, SERPINH1, PLOD2, PDGFA, ITGAV) and extracellular matrix (COL11A1, SPP1, COMP, BGN). Within-participant changes between baseline and post-treatment were assessed using the Wilcoxon signed rank test. Differences between treatment arms were assessed using the Wilcoxon rank sum test and the Kruskal-Wallis test. Results: endoxifen-gel at both doses incurred no dermal or systemic toxicity nor change in circulating biomarkers (IGF1, SHBG) compared to placebo. All treated breasts contained endoxifen at each of five sampling locations; the median per-person tissue concentration in the treated participants was 0.6 ng/g (IQR 0.4-1.6), significantly higher (p < 0.001) than the median plasma concentration (0.2 ng/mL, IQR 0.2-0.2). The median ratio of the more potent (Z)-isomer to (E)-isomer at each breast location was 1.5 (IQR 0.96-2.54, p < 0.05). We observed non-significant overall reduction in tumor Ki67 LI (median change -1.7 with IQR -5.8, 3.6) and gene expression of MKI67 and the genes associated with proliferation, and cell cycle. However, downregulation of invasion signature genes (FN1, SERPINH1, PLOD2, PDGFA, ITGAV) was significant (p=0.03) and a cluster of six participants demonstrated a consistent reduction of MKI67 and multiple gene signatures suggesting the possibility that their tumors might be early responders to low endoxifen exposure within 3 to 4 weeks of treatment. Conclusions: our evaluation of transdermal (E/Z) endoxifen 10 and 20 mg in hydroalcoholic/oleic acid formulation demonstrates lack of early dermal toxicity, and good tolerability over a month or so of therapy. Our findings warrant development of transdermal formulations of (Z)-endoxifen with better dermal permeation for a Phase 2 breast cancer prevention trial compared to low-dose oral SERMs so that women at high-risk for breast cancer may have a range of cancer prevention options available to them. Citation Format: Oukseub Lee, Latifa Bazzi, Yanfei Xu, Erik Pearson, Minhua Wang, Omid Hosseini, Azza Akasha, Jennifer Choi, Masha Kocherginsky, Kelly Benante, Thomas Helland, Gunnar Mellgren, Eileen Dimond, Marjorie Perloff, Brandy Heckman-Stoddard, Seema Khan, Scott Karlan, Melissa Pilewskie. A randomized Phase I pre-operative window trial of transdermal endoxifen in women planning mastectomy: evaluation of dermal safety, intra-mammary drug distribution, and biologic effects [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 RF01-02.

Abstract 3985: Immune-competent “NoGlow” transgenic mice overcome restrictive tolerance to multiple xenoantigens and simultaneously permit identification, tracking, and temporal gene expression in tumor cells

Abstract The ability to temporally regulate gene expression and track labeled cells make animal models powerful biomedical tools. However, sudden exposure to xenobiotic genes (e.g. GFP, luciferase (luc), or rtTA3) triggers inadvertent immune responses that can significantly suppress foreign gene expression or result in complete rejection of transplanted cells. Although germline expression of foreign genes somewhat addresses these challenges, native fluorescence and bioluminescence abrogates their utility as cellular reporters and spatiotemporally restricted expression can lead to suboptimal xenoantigen tolerance. Even in endogenous cancer models, we find that the means of tissue-directed expression of the same foreign gene yield significant differences in overall tolerance. Thus, the degree of tolerance to foreign genes strongly impacts the utility of different genetic modification and can strongly influence experimental outcomes and interpretations. To overcome these unwanted immune responses and enable reliable cell tracking/gene regulation, we developed a novel mouse model that selectively expresses antigen-intact but non-functional forms of GFP, luc, and rtTA3. These "NoGlow" mice possess no detectable background fluorescence or luminescence and mount limited adaptive immune responses against transgenic xenoantigens even after vaccination. Furthermore, we demonstrate that NoGlow mice allow tracking and tetracycline inducible gene regulation of triple-transgenic cells expressing GFP/luc/rtTA3, in contrast to transgene-negative immune competent mice which completely eliminate these cells. Notably, this model enables de novo metastasis from orthotopically implanted, triple-transgenic tumor cells, despite high xenoantigen expression. Finally, we observed model and sex-based differences in immune tolerance to the encoded xenoantigens after activation by different tissue-specific CREs, illustrating the obstacles of tolerizing animals to foreign genes and validating the utility of NoGlow mice to dissect the mechanisms of central and peripheral tolerance. Altogether, the NoGlow model provides a critical resource for in vivo studies across multiple disciplines, including oncology, developmental biology, infectious disease, autoimmunity, and transplantation. Citation Format: Timothy N. Trotter, Andrea Wilson, Jason McBane, Carina Dagotto, Xiao Yang, Junping Wei, Gangjun Lei, Hannah Thrash, H. Kim Lyerly, Zachary Hartman. Immune-competent “NoGlow” transgenic mice overcome restrictive tolerance to multiple xenoantigens and simultaneously permit identification, tracking, and temporal gene expression in tumor 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 3985.

Abstract 293: Distinctive roles of mammary adipomes in breast cancer progression: Insights from functional analysis and differential gene expression

Abstract Breast Cancer (BC) is a heterogeneous disease that impacts treatment response. This is due in part to the tumor microenvironment (TME), which contributes significantly to BC pathogenesis, including metastasis. Women with higher breast density and reduced breast fat face an increased BC risk across multiple tumor subtypes. This is exemplified by the correlation between increased tumor progression, the appearance of fibrosis and the loss of mature adipocytes. While the role of adipokines in this process has been studied extensively, the contribution of adipocyte-derived extracellular vesicles (EV) or "adipomes" in tumorigenesis and metastasis remains poorly understood. To address this question, we developed a novel technique for isolating intact adipomes from normal mammary fat (MF) obtained from wild-type C57BL/6 mice as well as from tumor-associated MF (TAMF) obtained from mice inoculated orthotopically with EO771 mammary tumor cells. Adipome isolation involved high-speed centrifugation to separate macro- and micro-vesicles, purification using the ExoQuick EV isolation system, and immunomagnetic capture of adipomes. Characterization of adipomes by transmission electron microscopy revealed two adipome populations, large L-adipomes of ~350 nm, and small S-adipomes of ~40 nm. Notably, the quantity of L-adipomes in tumor-associated mammary fat (TAMF) was 10-fold greater than the amount of L-adipomes in the MF of non-tumor-bearing mice. To identify possible functional roles of L-adipomes in the pathogenesis of BC, lipidomic profiling was undertaken. L-Adipomes from TAMF showed significantly altered lipid species in comparison to MF-derived L-adipomes. Adipomes were further characterized by treating EO771 cells for 72 hr with TAMF- or MF-derived adipomes at a cell to adipome ratio of 1:10, followed by RNAseq analysis. Differentially upregulated genes in TAMF-derived L-adipomes included Esr1, Erbb2, Tgfb1, Bcl2, MMP2, and Notch associated with fibrosis, EMT, angiogenesis and invasion and downregulation of genes linked to tumor suppression, apoptosis, and mitochondrial metabolism. Overall, our study illustrates the importance of tumor-associated adipomes in regulating gene expression in tumor cells, which emphasizes the specificity of adipome content relating to the cell-of-origin and the host's pathologic and metabolic state and in modulating signaling pathways involved in BC progression. Citation Format: Hariprasad Thangavel, Kezia Lizardo, Robert I. Glazer, Jyothi F. Nagajyothi. Distinctive roles of mammary adipomes in breast cancer progression: Insights from functional analysis and differential gene expression [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 293.

Abstract 2660: CXCL9/10 is critical for immunotherapeutic efficacy by recruiting and activating T lymphocytes in tumor microenvironment

Abstract Background: Tumor immunotherapies targeting PD-(L)1 exhibit anti-tumor efficacy in only about 20% of patients with a variety of cancers. Literature has demonstrated that “hot tumor” which contains high T lymphocytes in tumor microenvironment exhibits a better response to immunotherapies than “cold tumor”. Therefore, we hypothesize that T lymphocyte-recruited factors play the roles determining the immunotherapeutic efficacy, including CXCL9, 10, and 11, which are located in human chromosome 4 with consistent co-expression and protein structure. Aims: The aim of this study is to investigate whether CXCL9/10 is critical in determining the anti-PD-(L)1 immunotherapeutic efficacy. Methods: GEPIA, cBioPortal, and Kaplan-Meier Plotter were used to investigate the gene expression, co-expression, and immunotherapeutic association for CD274 (PD-L1), ICAM1, and CXCL9/10 in lung, colon, and liver cancers. qPCR and flow cytometry were used to detect the gene expression in tumor cells and T lymphocytes, particularly comparing the differences between LL/2 and CT26 cells whereas LL/2 is immunotherapy-resistant and CT26 is immunotherapy-sensitive. CD8+ T cell transwell assay and qPCR were used to measure the function of CXCL10 on CD8+ T cell migration and activation, whereas IFNγ and IL-2 as the activation markers. Results: We found that CXCL9/10 overexpressed in a variety of tumors, including lung, colon, and liver tumors with a correlation with PD-L1. However, PD-L1 was not overexpressed in most tumors. High PD-L1 and CXCL10 are basically associated with better survival rates in tumor patients receiving immunotherapies. Correlation analysis revealed that IRF-1 and STAT1 were correlated with PD-L1 and CXCL10 expression in lung cancer. Since literature has indicated that LL/2-derived syngeneic tumor exhibits immunotherapy-resistant and CT26 exhibits immunotherapy-sensitive, we further compared the gene expression between them. We found that IFNA, IFNG, CXCL10, and PD-L1 all expressed higher levels in CT26 cells than LL/2. Moreover, we found that T lymphocytes specifically expressed high CXCR3, a receptor of CXCL9/10, including CD4+ T and CD8+ T cells by flow cytometry. As expected, CD8+ T cells can be recruited in a transwell assay by IFNα and CXCL9. In addition, we found that IFNα induced CXCL10 expression in monocytes and CXCL10 increased activation markers IFNγ and IL-2 in CD4+ T and CD8+ T cells. Conclusions: This study revealed that CXCL9/10 expression is correlated with PD-L1 in tumors, resulting in better immunotherapeutic efficacy. The evidence demonstrated that CT26 intrinsically exhibited higher IFNs and CXCL9/10 compared to LL/2. Therefore, CXCL9/10 potentially recruits and activates CD8+ T cells to tumor microenvironment, transferring “cold tumor” to “hot tumor”. This study indicates that CXCL9/10 is critical for immunotherapeutic efficacy. Citation Format: Chun-Chia Cheng, Jungshan Chang, Zong-Lin Sie, Ai-Sheng Ho, Cheng-Liang Peng, Chun-Chao Chang. CXCL9/10 is critical for immunotherapeutic efficacy by recruiting and activating T lymphocytes in tumor microenvironment [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 2660.

Abstract 5493: Digital spatial profiling of MC38 colon carcinoma following checkpoint inhibition

Abstract Immunotherapy aims to bolster the immune response within the tumor microenvironment (TME). However, checkpoint inhibitors utilized in the treatment of colon carcinoma have demonstrated efficacy only in certain patients. To develop more effective therapeutics, strategies that navigate the heterogeneous nature of the TME must be employed. The MC38 tumor model, known for its immunologically active characteristics and responsiveness to certain immunotherapies, is widely used for preclinical drug development. Furthermore, elevated levels of pan-cytokeratin (PanCK) expression in tumors have shown an association with diminished survival rates among colon carcinoma patients. In this study, we used the GeoMx® DSP platform (NanoString Technologies) to investigate the spatial expression profile of PanCK in MC38 tumors and analyze the impact that PanCK expression exerts on anti-PD-1 induced gene expression in tumor and immune cells. To this end, C57BL/6J female mice were implanted with MC38 cells, enrolled into groups, and treated with anti-PD-1 antibody. Tumor growth was monitored by caliper measurements and anti-PD-1 produced minimal responses with a Day 22 median % Delta T/C value of 88%. For DSP analysis, tumors were collected 10 days post-treatment, formalin fixed for paraffin embedding, and then sectioned for PanCK and CD45 protein visualization. Regions of interest were drawn to create a matrix analysis that defined regions with high or low PanCK expression, that were proximal or distal to CD45+ immune rich regions of the tumor. A segmentation algorithm was applied to examine gene expression within each region using the GeoMx Mouse Whole Transcriptome Atlas. Cluster analysis revealed an enrichment of apoptosis and autophagy gene sets in tumor cells that were proximal to immune rich regions. In the CD45+ segment, immune cells adjacent to high PanCK-expressing cells contained 318 genes with significantly increased expression levels when compared to low PanCK-expressing cells, and only 11 genes with decreased expression levels. The upregulated genes were enriched for numerous biomarkers of T cell recruitment and activation, including cd3, cd8, cxcr3, ccl5, cxcl9, icos, and cd27. Next, we conducted a linear mixed model analysis to examine the impact of PanCK expression on anti-PD-1 induced gene regulation. Notably, an enrichment of genes that code for T cell exhaustion biomarkers was observed in immune cells near PanCK high expressing cells, including ctla4, slamf7, tigit, and lag3. In summary, the most pronounced transcriptomic changes occurred within immune cells situated near tumor cells displaying high PanCK expression and suggest that PanCK expression correlates directly with T cell recruitment and activation but also exhaustion. Furthermore, these data emphasize the significance of spatial analysis as a valuable component of preclinical research, as demonstrated in MC38 tumor model. Citation Format: Priyanka Singh, Daniel Saims, Sheri Barnes, David Draper. Digital spatial profiling of MC38 colon carcinoma following checkpoint inhibition [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 5493.

Abstract 1577: Cancer associated fibroblast - tumor cell crosstalk enhances epithelial to mesenchymal transition and promotes a classical to basal switch in human pancreatic ductal adenocarcinoma

Abstract Introduction: Cancer associated fibroblasts (CAF) drive a complex tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) through incompletely understood interactions. Two major CAF subtypes, myofibroblastic (myCAF) and inflammatory (iCAF), dominate the tumor landscape and have been defined by gene expression profiles. There is significant plasticity between subtypes and alterations in CAF behavior likely drive changes in tumorigenicity and immune evasion. Additionally, PDAC tumor cells usually present with a predominant classical or basal phenotype that is associated with clinical outcomes. We hypothesized that CAF phenotype impacts tumor cell gene expression and alters the clinically-relevant tumor subtyping. Methods: We interrogated cellular crosstalk in the PDAC TME using a novel three-dimensional, patient-matched coculture system of patient-derived organoids (PDO) and CAFs, established from 12 patients undergoing surgical resection. Molecular characterization included bulk RNA sequencing, proteome profiling (107 factor), flow cytometry, multiplex RNA Scope, imaging mass cytometry (IMC), and RT-qPCR. Results: PDO gene expression analysis from bulk RNAseq demonstrated CAF coculture induced phenotypic shifts in the Moffitt subtype from a classical to a basal phenotype. Further, CAF coculture enhanced epithelial to mesenchymal transition (EMT) across all patients, even those with a baseline basal phenotype. We hypothesized this could be due to both cell-cell crosstalk and secreted factors. We therefore treated PDO with CAF-conditioned media (CM) and demonstrated that CM is sufficient to drive the observed classical-to-basal shift by RT-qPCR. Secretome analysis of CM derived from 3 CAF lines identified HGF, FGF19, and IL-8 as factors contributing to this transcriptional change. Next, we used spatial - omics (transcriptomics and proteomics) to examine the validity of these findings in source patient tissue. Using RNA Scope, we discovered distinct tumor neighborhoods expressing basal mRNAs KRT17 and KRT6a surrounded by CAFs compared to those expressing classical genes TFF1 and GATA6. We then used IMC to further explore CAF subtypes in patient tumors and found FAP+ myCAF regions corresponded to basal tumor regions, while CXCL12+ iCAFs were found in classical regions. Conclusions: PDAC tumor biology is likely driven by interactions between cancer cells and CAFs. Potential mediators of these interactions include both cell-cell contact and secreted factors. We introduce a novel approach combining in vitro coculture with tissue-based assays, demonstrating that modern culture techniques preserve cell type heterogeneity and plasticity. Together, these data demonstrate that a CAF-rich TME drives tumor cell plasticity not only towards EMT but also a more basal disease phenotype. Citation Format: Samantha Guinn, Brayan Perez, Jignasha Patel, Jae Lee, Daniel Zabransky, Won Ho, Elana J. Fertig, Emma Kartalia, Joseph Tandurella, Richard Burkhart, Jacquelyn W. Zimmerman, Elizabeth Jaffee. Cancer associated fibroblast - tumor cell crosstalk enhances epithelial to mesenchymal transition and promotes a classical to basal switch in human pancreatic ductal adenocarcinoma [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 1577.

Increased stiffness of extracellular matrix enhanced chemoresistance in 3D-bioprinted ovarian cancer model

Ovarian cancer is a gynecological malignancy with a high mortality rate. The ovarian cancer microenvironment is a crucial factor affecting the overall and progression-free survival rates of patients with ovarian cancer. The biophysical factors of the tumor microenvironment, such as stiffness, can affect the gene expression and behavior of tumor cells. In this study, we utilized 3D bioprinting technology to construct ovarian cancer tumor models with varying levels of stiffness in vitro to investigate the effect of extracellular matrix stiffness on drug resistance of tumor cells. Our findings indicate that increasing the stiffness of extracellular matrix can attenuate the sensitivity of tumor cells to chemotherapeutic agents. Additionally, the increased stiffness of 3D tumor model may promote malignant phenotypes, such as tumor stemness and tumor progression.

Influences of probiotics and gut microbiota on immunomodulation for the treatment of patients with cancer: a systematic review

Introduction: Immunomodulation and immunostimulation are the main mechanisms of action of probiotics in the fight against cancer. Objective: It was to carry out a systematic review of the influences of probiotics and gut microbiota on immunomodulation for the treatment of cancer patients. Methods: The PRISMA Platform systematic review rules were followed. The research was carried out from September to October 2023 in the Web of Science, Scopus, PubMed, Science Direct, Scielo, and Google Scholar databases. The quality of the studies was based on the GRADE instrument and the risk of bias was analyzed according to the Cochrane instrument. Results and Conclusion: A total of 125 articles were found, and 35 articles were evaluated in full and 18 were included and developed in the present systematic review study. Considering the Cochrane tool for risk of bias, the overall assessment resulted in 45 studies with a high risk of bias and 15 studies that did not meet GRADE and AMSTAR-2. Most studies showed homogeneity in their results, with X2=55.4%>50%. It was concluded that probiotics suppress inflammation by inhibiting several signaling pathways and reducing the global activation of NF-κβ and the production of pro-inflammatory cytokines. Probiotics influence receptor antagonism, receptor expression, binding and expression of adapter proteins, expression of negative regulatory signal molecules, and mainly the induction of microRNAs. These microRNAs can modulate the gene expression of tumor cells together with regulatory T cells that have important functions in the tumor microenvironment, mainly in inducing immune evasion. Some modulatory effects of probiotics include the production of cytokines by epithelial cells, increased mucin secretion and phagocytosis and NK cell activity, activation of T and NKT cells, stimulation of IgA production, and decreased proliferation of T cells. The gut microbiota has a major impact on the systemic immune system. The specific microbiota controls cell differentiation in which CD4+ T cells (Th17 cells) secrete IL-17. The presence of Th17 cells and regulatory T cells is associated with the gut microbiota.

TIE1 promotes cervical cancer progression via Basigin-matrix metalloproteinase axis.

Background: Tyrosine kinase with immunoglobulin and EGF-like domains 1 (TIE1) is known as an orphan receptor prominently expressed in endothelial cells and participates in angiogenesis by regulating TIE2 activity. Our previous study demonstrated elevated TIE1 expression in cervical cancer cells. However, the role of TIE1 in cervical cancer progression, metastasis and treatment remains elusive. Methods: Immunohistochemistry staining for TIE1 and Basigin was performed in 135 human cervical cancer tissues. Overexpressing vectors and siRNAs were used to manipulate gene expression in tumor cells. Colony formation, wound healing, and transwell assays were used to assess cervical cancer cell proliferation and migration in vitro. Subcutaneous xenograft tumor and lung metastasis mouse models were established to examine tumor growth and metastasis. Co-Immunoprecipitation and Mass Spectrometry were applied to explore the proteins binding to TIE1. Immunoprecipitation and immunofluorescence staining were used to verify the interaction between TIE1 and Basigin. Cycloheximide chase assay and MG132 treatment were conducted to analyze protein stability. Results: High TIE1 expression was associated with poor survival in cervical cancer patients. TIE1 overexpression promoted the proliferation, migration and invasion of cervical cancer cells in vitro, as well as tumor growth and metastasis in vivo. In addition, Basigin, a transmembrane glycoprotein, was identified as a TIE1 binding protein, suggesting a pivotal role in matrix metalloproteinase regulation, angiogenesis, cell adhesion, and immune responses. Knockdown of Basigin or treatment with the Basigin inhibitor AC-73 reversed the tumor-promoting effect of TIE1 in vitro and in vivo. Furthermore, we found that TIE1 was able to interact with and stabilize the Basigin protein and stimulate the Basigin-matrix metalloproteinase axis. Conclusion: TIE1 expression in cervical cells exerts a tumor-promoting effect, which is at least in part dependent on its interaction with Basigin. These findings have revealed a TIE2-independent mechanism of TIE1, which may provide a new biomarker for cervical cancer progression, and a potential therapeutic target for the treatment of cervical cancer patients.

Spatial transcriptomic interrogation of the tumour-stroma boundary in a 3D engineered model of ameloblastoma

Stromal cells are key components of the tumour microenvironment (TME) and their incorporation into 3D engineered tumour-stroma models is essential for tumour mimicry. By engineering tumouroids with distinct tumour and stromal compartments, it has been possible to identify how gene expression of tumour cells is altered and influenced by the presence of different stromal cells. Ameloblastoma is a benign epithelial tumour of the jawbone. In engineered, multi-compartment tumouroids spatial transcriptomics revealed an upregulation of oncogenes in the ameloblastoma transcriptome where osteoblasts were present in the stromal compartment (bone stroma). Where a gingival fibroblast stroma was engineered, the ameloblastoma tumour transcriptome revealed increased matrix remodelling genes. This study provides evidence to show the stromal-specific effect on tumour behaviour and illustrates the importance of engineering biologically relevant stroma for engineered tumour models. Our novel results show that an engineered fibroblast stroma causes the upregulation of matrix remodelling genes in ameloblastoma which directly correlates to measured invasion in the model. In contrast the presence of a bone stroma increases the expression of oncogenes by ameloblastoma cells.

The Role of microRNAs in Gene Expression and Signaling Response of Tumor Cells to an Acidic Environment

Many tumors are characterized by marked extracellular acidosis due to increased glycolytic metabolism, which affects gene expression and thereby tumor biological behavior. At the same time, acidosis leads to altered expression of several microRNAs (Mir7, Mir183, Mir203, Mir215). The aim of this study was to analyze whether the acidosis-induced changes in cytokines and tumor-related genes are mediated via pH-sensitive microRNAs. Therefore, the expression of Il6, Nos2, Ccl2, Spp1, Tnf, Acat2, Aox1, Crem, Gls2, Per3, Pink1, Txnip, and Ypel3 was examined in acidosis upon simultaneous transfection with microRNA mimics or antagomirs in two tumor lines in vitro and in vivo. In addition, it was investigated whether microRNA expression in acidosis is affected via known pH-sensitive signaling pathways (MAPK, PKC, PI3K), via ROS, or via altered intracellular Ca2+ concentration. pH-dependent microRNAs were shown to play only a minor role in modulating gene expression. Individual genes (e.g., Ccl2, Txnip, Ypel3) appear to be affected by Mir183, Mir203, or Mir215 in acidosis, but these effects are cell line-specific. When examining whether acid-dependent signaling affects microRNA expression, it was found that Mir203 was modulated by MAPK and ROS, Mir7 was affected by PKC, and Mir215 was dependent on the intracellular Ca2+ concentration. Mir183 could be increased by ROS scavenging. These correlations could possibly result in new therapeutic approaches for acidotic tumors.

Important Role for Glutamine in Human and Mouse TME Models of CLL ; Towards Development of a PET Tracer As a Novel Diagnostic Tool

The amino acid transporter ASCT2, encoded by the gene SLC1A5, is the main glutamine importer in proliferating human and mouse cells. We recently showed that glutamine is the main fuel of the tricarboxylic acid (TCA) cycle in cells from chronic lymphocytic leukemia (CLL) patients, and that blocking its import attenuated CD40/BCR-induced venetoclax (VEN) resistance 1. In the current study, we have investigated the role of ASCT2 and the consequences of its inhibition in human and mouse CLL cells and in healthy lymphocytes by flow cytometry, gene expression analysis, extracellular flux analysis, metabolomics, CRISPR/Cas9 gene editing and adoptive-cell transfer experiments. To enable in vivo assessment of ASCT2 in the future with Positron Emission Tomography (PET), we have explored the potential use of 18F-labelelled ( 2S,2R)-4-fluoroglutamine (4- 18F]FGln) 2 as PET tracer. Increased expression of ASCT2 upon PMA/ionomycin stimulation was observed in healthy B and T cells. In BCR/CD40-stimulated CLL cells, the increased expression was irrespective of IGHV status. We dissected the main routes of glutamine usage in CD40- and BCR-stimulated CLL cells and found that in addition to its role in the TCA cycle, glutamine import is linked to mTOR signaling. The ASCT2 inhibitor V9302 impaired mTOR signaling and decreased translation of new proteins, including the anti-apoptotic proteins Bcl-XL and Mcl-1. Of relevance for therapeutic approaches, the effector and proliferative capacity of human T cells was preserved upon V9302 treatment. This suggests that blocking ASCT2 will not affect immune responses. The SLC1A5 gene was deleted by CRISPR/Cas9 technique in murine TCL1 cells 3. Adoptive cell transfer experiments of SLC1A5-KO and WT TCL1 cells injected at 50-50% ratio in recipient mice showed selective outgrowth of SLC1A5-WT cells, with no detection of the SLC1A5-KO in the spleen of overt leukemic mice. This indicates that TCL1 leukemic cells rely on SLC1A5/ASCT2 for growth. Finally, we explored the high expression of ASCT2 and glutamine addiction in CLL from the diagnostic perspective. With the aim of improving the currently suboptimal [ 18F]FDG PET for imaging of CLL LN sites 4, we successfully synthesized 4-[ 18F]FGln. We observed uptake in CD40-stimulated CLL cells, which was decreased in the presence of V9302. Experiments to perform PET with 4-[ 18F]FGln in TCL1 mice to demonstrate an in vivo proof of concept are ongoing. Overall, these data support a crucial role for ASCT2 and glutamine in leukemia development and VEN resistance in CLL. This provides a basis for targeting the transporter for therapeutic options and/or taking advantage of its function for diagnostic approaches using 4-[ 18F]FGln as PET tracer. 1. Chen, Z. et al. Characterization of metabolic alterations of chronic lymphocytic leukemia in the lymph node microenvironment. Blood 140, 630-643, doi:10.1182/blood.2021013990 (2022). 2. Qu, W. et al. Synthesis of optically pure 4-fluoro-glutamines as potential metabolic imaging agents for tumors. J Am Chem Soc 133, 1122-1133, doi:10.1021/ja109203d (2011). 3. Nardi, F. et al. Assessing gene function in human B cells: CRISPR/Cas9-based gene editing and mRNA-based gene expression in healthy and tumor cells. Eur J Immunol 52, 1362-1365, doi:10.1002/eji.202149784 (2022). 4. Rhodes, J. M. & Mato, A. R. PET/Computed Tomography in Chronic Lymphocytic Leukemia and Richter Transformation. PET Clin 14, 405-410, doi:10.1016/j.cpet.2019.03.007 (2019).

SCAR: Single-cell and Spatially-resolved Cancer Resources.

Advances in sequencing and imaging technologies offer a unique opportunity to unravel cell heterogeneity and develop new immunotherapy strategies for cancer research. There is an urgent need for a resource that effectively integrates a vast amount of transcriptomic profiling data to comprehensively explore cancer tissue heterogeneity and the tumor microenvironment. In this context, we developed the Single-cell and Spatially-resolved Cancer Resources (SCAR) database, a combined tumor spatial and single-cell transcriptomic platform, which is freely accessible at http://8.142.154.29/SCAR2023 or http://scaratlas.com. SCAR contains spatial transcriptomicdata from 21 tumor tissues and single-cell transcriptomic data from 11 301 352 cells encompassing 395 cancer subtypes and covering a wide variety oftissues, organoids, and cell lines. This resource offers diverse functional modules to address key cancer research questions at multiple levels, including the screening of tumor cell types, metabolic features, cell communication and gene expression patterns within the tumor microenvironment. Moreover, SCAR enables the analysis of biomarker expression patterns and cell developmental trajectories. SCAR also provides a comprehensive analysis of multi-dimensional datasets based on 34 state-of-the-art omics techniques, serving as an essential tool for in-depth mining and understanding of cell heterogeneity and spatial location. The implications of this resource extend to both cancer biology research and cancer immunotherapy development.

Systemic Delivery of Magnetogene Nanoparticle Vector for Gene Expression in Hypoxic Tumors.

Cancer is a disease that causes millions of deaths per year worldwide because conventional treatments have disadvantages such as unspecific tumor selectivity and unwanted toxicity. Most human solid tumors present hypoxic microenvironments and this promotes multidrug resistance. In this study, we present "Magnetogene nanoparticle vector" which takes advantage of the hypoxic microenvironment of solid tumors to increase selective gene expression in tumor cells and reduce unwanted toxicity in healthy cells; this vector was guided by a magnet to the tumor tissue. Magnetic nanoparticles (MNPs), chitosan (CS), and the pHRE-Luc plasmid with a hypoxia-inducible promoter were used to synthesize the vector called "Magnetogene nanoparticles" by ionic gelation. The hypoxic functionality of Magnetogene vector nanoparticles was confirmed in the B16F10 cell line by measuring the expression of the luciferase reporter gene under hypoxic and normoxic conditions. Also, the efficiency of the Magnetogene vector was confirmed in vivo. Magnetogene was administered by intravenous injection (IV) in the tail vein and directed through an external magnetic field at the site of tumor growth in C57Bl/6 mice. A Magnetogene vector with a size of 50 to 70 nm was directed and retained at the tumor area and gene expression was higher at the tumor site than in the others tissues, confirming the selectivity of this vector towards hypoxic tumor areas. This nanosystem, that we called the "Magnetogene vector" for systemic delivery and specific gene expression in hypoxic tumors controlled by an external magnetic designed to target hypoxic regions of tumors, can be used for cancer-specific gene therapies.

HGG-18. SINGLE CELL RNA-SEQ ANALYSIS OF PEDIATRIC HIGH-GRADE GLIOMA PATIENT SAMPLES IDENTIFIES COMMON GLIAL AND IMMUNE CELL TYPES THAT HELP TO EXPLAIN HETEROGENEITY AND TUMORIGENESIS

Abstract BACKGROUND Pediatric high-grade gliomas (PHGG) are aggressive, undifferentiated central nervous system (CNS) tumors. PHGG comprises subtypes that differ phenotypically, histologically and by cellular composition. Despite intensive research, overall survival rates have remained for decades around 2% for diffuse midline glioma (DMG) and 20% for non-DMG PHGG. We hypothesized that PHGG’s heterogeneity, a key difficulty in devising therapies, results from functional and lineage differences in tumor cell types. METHODS Using 19 PHGG samples from our institution’s pediatric brain tumor bank, we constructed a single-cell RNA-Seq (scRNA-Seq) dataset that included tumor and immune cells, generated predicted cell types by mapping the scRNA-Seq data onto known cell types, and performed differential gene expression and geneset enrichment analyses. We acquired bulk RNA-Seq and DNA methylation data to characterize overall tumor properties. RESULTS Samples consisted of several principal tumor cell types, including cells with astrocyte characteristics, oligodendrocyte progenitor cell (OPC) characteristics, and cells that expressed OPC markers but had slight enrichment in mesenchymal and inflammatory gene expression (OPC-like/Mes cells). We also found a potential stemlike population that expressed neural stem cell markers. The predicted astrocytes, OPCs and OPC-like/Mes cells differed in their gene expression profiles, pathway enrichment, and tendency to proliferate as assessed from expression of cell cycle genes. A combined microglia/macrophage population, strongly enriched in mesenchymal and inflammatory gene expression, may serve to induce inflammatory gene expression in tumor cells. Key conclusions from the scRNA-Seq analysis were validated using multi-channel immunofluorescence staining. CONCLUSIONS PHGG comprises a small stemlike population and varying proportions of proliferating glial lineage tumor cells. CNS-resident microglia/macrophages may trigger mesenchymal gene expression in PHGG. We will use our results to investigate the roles of PHGG’s diverse tumor cell populations in resistance to radiotherapy and to develop treatments to target individual cell types comprising PHGG.

An information theoretic approach to detecting spatially varying genes

A key step in spatial transcriptomics is identifying genes with spatially varying expression patterns. We adopt an information theoretic perspective to this problem by equating the degree of spatial coherence with the Jensen-Shannon divergence between pairs of nearby cells and pairs of distant cells. To avoid the notoriously difficult problem of estimating information theoretic divergences, we use modern approximation techniques to implement a computationally efficient algorithm designed to scale with in situ spatial transcriptomics technologies. In addition to being highly scalable, we show that our method, which we call maximization of spatial information (Maxspin), improves accuracy across several spatial transcriptomics platforms and a variety of simulations when compared with a variety of state-of-the-art methods. To further demonstrate the method, we generated in situ spatial transcriptomics data in a renal cell carcinoma sample using the CosMx Spatial Molecular Imager and used Maxspin to reveal novel spatial patterns of tumor cell gene expression.

Phenotypic and genotypic infidelity in B-lineage neoplasms, including transdifferentiation following targeted therapy: Report from the 2021 SH/EAHP Workshop.

Session 2 of the 2021 Society for Hematopathology and European Association for Haematopathology Workshop collected examples of lineage infidelity and transdifferentiation in B-lineage neoplasms, including after targeted therapy. Twenty cases were submitted. Whole-exome sequencing and genome-wide RNA expression analysis were available on a limited subsample. A diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) was rendered on at least 1 biopsy from 13 patients. There was 1 case of acute myeloid leukemia (AML); the remaining 6 cases were mature B-cell neoplasms. Targeted therapy was administered in 7 cases of B-ALL and 4 cases of mature B-cell neoplasms. Six cases of B-ALL underwent lineage switch to AML or mixed-phenotype acute leukemia at relapse, 5 of which had rearranged KMT2A. Changes in maturational state without lineage switch were observed in 2 cases. Examples of de novo aberrant T-cell antigen expression (n = 2) were seen among the mature B-cell lymphoma cohort, and their presence correlated with alterations in tumor cell gene expression patterns. This cohort of cases enabled us to illustrate, discuss, and review current concepts of lineage switch and aberrant antigen expression in a variety of B-cell neoplasms and draw attention to the role targeted therapies may have in predisposing neoplasms to transdifferentiation as well as other, less expected changes in maturational status.

Abstract 4332: Characterizing cancer-associated fibroblasts in prostate cancer using Vizgen’s MERSCOPETM Platform

Abstract Understanding the spatial complexities within cancer provides crucial information for evaluating individual tumor development and progression; recent research in prostate cancer has shown how critical it is for oncologists to maintain a spatial context while characterizing a highly heterogeneous tumor microenvironment. As more and more research groups acknowledge the need for spatial biology solutions to solve difficult questions surrounding cell identity, heterogeneity, and interaction, Vizgen’s MERSCOPETM Platform provides a powerful tool to illuminate spatial information. Built on Multiplexed Error-Robust Fluorescence in situ Hybridization (MERFISH) technology, Vizgen’s MERSCOPETM enables the direct profiling of the spatial organization of intact tissue with subcellular resolution. Here, we use a 500-gene panel to demonstrate the MERSCOPE’s spatial capability, assessing the canonical signaling pathways of cancer, cancer type-specific genes, select immune genes, proto-oncogenes, and tumor-suppressor genes in patient-derived prostate cancer samples. Using the Vizgen Cell Boundary Stain kit, we were able to generate a molecular and cellular atlas of these individual patient tumors by clustering cells based on gene expression and mapping these clusters back onto a spatial representation of the tumor. We found specific clusters relating to prognostic outcomes in cancer-associated fibroblasts (CAFs) and myofibroblasts. Subsequently, we used single-cell RNA-seq datasets to impute related transcriptome data for these various fibroblast clusters to help characterize them more specifically. To understand the heterogeneity intrinsic to CAFs, we performed a neighborhood analysis to determine how proximity to immune cells, tumor cells, and other stromal cells altered gene expression in different CAF clusters. We found gene expression changes associated with inflammation and reactive stromal signaling in CAFs based on their proximity to tumor versus stromal tissue compartments. We also compared the gene expression of tumor cells near CAFs to those more deeply embedded within the tumor compartment. This revealed interesting transcriptional covariations between superficial layers of tumor and their surrounding stromal milieu involved in cell proliferation and signaling. These analyses demonstrate the transcriptional variation possible across fibroblastic stromal cells acting in tumor-adjacent or stromal contexts. Notably, we were able to map and catalogue different patterns of gene expression relating to inflammation, tumor proliferation, and reactive stromal signaling. These findings demonstrate how the MERSCOPE platform can provide deep insights into the complex heterogeneity observed in the tumor microenvironment. Citation Format: Ben Patterson, Cheng-Yi Chen, Nicolas Fernandez, Simran Kaushal, Leiam Colbert, Jiang He. Characterizing cancer-associated fibroblasts in prostate cancer using Vizgen’s MERSCOPETM Platform. [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 4332.

Abstract 5148: Interrogating immuno-oncological interactions in the tumor microenvironment

Abstract Single-cell RNA sequencing is a powerful and innovative tool for assessing individual cancer prognoses and outcomes; however, the process of generating this data typically destroys the spatial complexity of the tumor microenvironment. Understanding the spatial complexities within cancer provides crucial information for evaluating individual tumor development and progression. Vizgen’s MERSCOPETM Platform, built on Multiplexed Error-Robust Fluorescence in situ Hybridization (MERFISH) technology, enables the direct profiling of the spatial organization of intact tissue with subcellular resolution. Here, we present a Pan-Cancer approach to demonstrate the MERSCOPE’s ability to characterize breast, colon, prostate, ovarian, lung, and skin cancers from human clinical samples. Specifically, using a 500-gene panel to assess the canonical signaling pathways of cancer, cancer type-specific genes, select immune genes, proto-oncogenes, and tumor-suppressor genes, we show that the MERSCOPE was able to spatially profile gene expression across multiple tumor types. We utilized the Vizgen Cell Boundary Stain kit to generate a molecular and cellular atlas of individual patient tumors by clustering cells based on gene expression and mapping their spatial organization. To gather insight into immuno-oncological interactions and their effect on gene expression across these tumor types, we performed a neighborhood analysis to determine how proximity to immune cells altered gene expression in tumor cells, and vice versa. We found significant gene expression changes in MHC activation and downstream pathways in immune cells located nearest to tumor cells; in tumor cells nearest to immune cells, we identified increased expression of genes related to immune response mediation and inflammation. Furthermore, we compared the annotated immune cell populations across the various cancer types to observe differences in gene expression. Here, we used publicly available single-cell RNA-sequencing data to impute gene expression for these immune cell populations. This analysis demonstrated the transcriptional variation possible in identical immune cell types that are acting in different tissue contexts. Notably, we were able to map and catalogue different patterns of gene expression relating to inflammation, immune signaling, and immune cell exhaustion. These findings demonstrate how the MERSCOPE platform can provide deep insights into the complex heterogeneity observed between different tumor types and tissues. Citation Format: Ben Patterson, g-Yi Chen, Nicolas Fernandez, Leiam Colbert, Jiang He. Interrogating immuno-oncological interactions in the tumor microenvironment. [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 5148.