Stages Of Tumor Progression Research Articles

Assessing the metastatic potential of circulating tumor cells using an organ-on-chip model.

Metastatic lung cancer remains a leading cause of death worldwide, with its intricate metastatic cascade posing significant challenges to researchers and clinicians. Despite substantial progress in understanding this cascade, many aspects remain elusive. Microfluidic-based vasculature-on-chip models have emerged as powerful tools in cancer research, enabling the simulation of specific stages of tumor progression. In this study, we investigate the extravasation behaviors of A549 lung cancer cell subpopulations, revealing distinct differences based on their phenotypes. Our results show that holoclones, which exhibit an epithelial phenotype, do not undergo extravasation. In contrast, paraclones, characterized by a mesenchymal phenotype, demonstrate a notable capacity for extravasation. Furthermore, we observed that paraclones migrate significantly faster than holoclones within the microfluidic model. Importantly, we found that the depletion of vascular endothelial growth factor (VEGF) effectively inhibits the extravasation of paraclones. These findings highlight the utility of microfluidic-based models in replicating key aspects of the metastatic cascade. The insights gained from this study underscore the potential of these models to advance precision medicine by facilitating the assessment of patient-specific cancer cell dynamics and drug responses. This approach could lead to improved strategies for predicting metastatic risk and tailoring personalized cancer therapies, potentially involving the sampling of cancer cells from patients during tumor resection or biopsies.

7092 Blocking of Aggressive Tumor Progression of Anaplastic Thyroid Cancer by p53

Abstract Disclosure: E. Hwang: None. R. Chari: None. T. Kimura: None. S. Cheng: None. Background: Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancer, with very limited treatment options. Patients succumb to death within 6-12 months after diagnosis. It has been observed that mutations of p53, occurring as tumorigenesis progresses, is associated with the aggressiveness of ATC. The present studies tested the hypothesis that wild type p53 (WTp53) could block tumor progression and mitigate its aggressiveness. Methods: We used human 8505C cells as a model, derived from human ATC tumors, harboring BRAFV600E mutation and one allele of mutated p53C742G. We re-expressed WTp53 or mutant p53C742G into 8505C cells (8505C-WTp53 or 8505C-MTp53 cells, respectively) via lentiviral transduction. We analyzed the effects of exogenous expressed WTp53 and mutant p53 on cell proliferation, apoptosis, and migration in vitro and the progression of tumors in vivo mouse xenograft models. Results: We showed that p53 proteins were more abundantly expressed in 8505C-WTp53 or 8505C-MTp53 cells than the parental 8505C cells. Using WTp53 luciferase reporter (PG-13), we demonstrated that the expressed WTp53 was functional as the transcription activity of p53 was higher than the parental 8505C cells. Consistently, in 8505C-MTp53, the reporter activity of PG-13 was not detected. The expressed WTp53 inhibited cell proliferation and decreased cell migration. Immunohistochemical analysis (IHC) showed decreased proliferation marker Ki67 and increased cleaved caspase-3, indicating the induction of apoptosis in 8505C-WTp53 cells. Importantly, we found that the NIS expression was increased in 8505C-WTp53 cells, but not in 8505C-MTp53 cells, as compared with 8505C cells. Xenograft studies showed that tumor progression was inhibited in nude mice injected with 8505C-WTp53 cells as compared with tumors induced by 8505C parental cells and 8505C-MTp53 cells. Consistent with in vitro findings, IHC demonstrated decreased proliferation marker Ki67 and increased cleaved caspase-3 proteins in tumors induced by 8505C-WTp53 cells. Furthermore, the expression of apoptotic regulators such as BAX and PUMA, known as p53 target genes, were elevated in 8505C-WTp53 cultured cells and in 8505C-WTp53-induced tumors, indicating induction of apoptosis. Conclusions: Our findings showed that the aggressiveness of ATC, caused by mutant p53 in late stage of tumor progression, can be mitigated by exogenous expression of WTp53. These results provided new insights into the deleterious actions of mutation of p53 in ATC tumor development. Importantly, our studies revealed that targeting p53 in ATC could be considered as a potential therapeutic opportunity. Presentation: 6/3/2024

Identifying targetable metabolic dependencies across colorectal cancer progression

Colorectal cancer (CRC) is a multi-stage process initiated through the formation of a benign adenoma, progressing to an invasive carcinoma and finally metastatic spread. Tumour cells must adapt their metabolism to support the energetic and biosynthetic demands associated with disease progression. As such, targeting cancer cell metabolism is a promising therapeutic avenue in CRC. However, to identify tractable nodes of metabolic vulnerability specific to CRC stage, we must understand how metabolism changes during CRC development. Here, we use a unique model system – comprising human early adenoma to late adenocarcinoma. We show that adenoma cells transition to elevated glycolysis at the early stages of tumour progression but maintain oxidative metabolism. Progressed adenocarcinoma cells rely more on glutamine-derived carbon to fuel the TCA cycle, whereas glycolysis and TCA cycle activity remain tightly coupled in early adenoma cells. Adenocarcinoma cells are more flexible with respect to fuel source, enabling them to proliferate in nutrient-poor environments. Despite this plasticity, we identify asparagine (ASN) synthesis as a node of metabolic vulnerability in late-stage adenocarcinoma cells. We show that loss of asparagine synthetase (ASNS) blocks their proliferation, whereas early adenoma cells are largely resistant to ASN deprivation. Mechanistically, we show that late-stage adenocarcinoma cells are dependent on ASNS to support mTORC1 signalling and maximal glycolytic and oxidative capacity. Resistance to ASNS loss in early adenoma cells is likely due to a feedback loop, absent in late-stage cells, allowing them to sense and regulate ASN levels and supplement ASN by autophagy. Together, our study defines metabolic changes during CRC development and highlights ASN synthesis as a targetable metabolic vulnerability in later stage disease.

Preoperative thromboelastography parameters in predicting the tumour stage of oral squamous cell carcinoma

BackgroundA variety of solid tumours, including oral squamous cell carcinoma (OSCC), can cause coagulation abnormalities, and this phenomenon is known as tumour-associated hypercoagulation. We aimed to explore the preoperative thromboelastography (TEG) parameter profiles of OSCC patients, and to investigate their trends in relation to tumour stage progression, and to evaluate their value for predicting cervical lymph node metastasis.MethodsData on thromboelastographic parameters and conventional coagulation indices were retrospectively collected, and comparisons were performed among preoperative primary OSCC patients (n = 311), recurrent/metastatic OSCC patients (n = 44) and a control group (n = 71). Among primary OSCC patients, the correlation with tumour stage and the predictive role of cervical lymph node metastasis were analyzed.ResultsHypercoagulability occurred in OSCC patients and tended to become more pronounced as the tumour progressed. The whole-time phase of coagulation increased with increasing T stage, while the early phase of coagulation increased with increasing N stage.ConclusionsPreoperative TEG parameters are closely related to tumour stage and progression, suggesting that TEG can be used as an important indicator for predicting tumour stage and as a potential biomarker.

MicroRNA: A Signature for the Clinical Progression of Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is the most common human leukemia. The disease is caused by abnormal proliferation and development of lymphocytes and their precursors in the blood and bone marrow (BM). Recent studies have shown that the CLL’s clinical course and outcome depend not only on genetic but also epigenetic factors. MicroRNAs (miRNAs) are involved in the development of hematological tumors, including CLL. The aim of this study is to identify the miRNA expression profile in CLL and determine the role of miRNAs in biological pathways associated with leukemogenesis in CLL. The following samples were used in this study: (1) samples obtained by sternal puncture and aspiration biopsy of BM (n = 115). They included samples from 21 CLL patients with anemia and indications for therapy and 45 CLL patients without anemia and with indications for therapy. The control group for the CLL BM samples consisted of patients with non-cancerous blood diseases (n = 35). (2) Lymph node (LN) samples (n = 20) were collected from CLL patients. The control group for the CLL LN samples consisted of patients with lymphadenopathy (n = 37). All cases were patients before treatment. We demonstrated a significant upregulation of miRNA-34a and miRNA-150 in CLL BM samples (p < 0.05) and downregulation of miRNA-451a in CLL LN samples (p < 0.05). We noted a dynamic increase in the levels of miRNA-150 and miRNA-34a in BM at various stages of tumor progression of CLL. We concluded that a dynamic picture of clinical manifestations of CLL closely correlates with changes in epigenetic characteristics of the tumor. Progression of the lymphoproliferative process and indications for cytoreductive therapy are associated with changes in the miRNA profile generated by cancer cells in different sites of clonal expansion.

Identification of oxidative stress signatures of lung adenocarcinoma and prediction of patient prognosis or treatment response with single-cell RNA sequencing and bulk RNA sequencing data

Lung adenocarcinoma (LUAD), the most common subtype of lung cancer globally, has seen improved prognosis with advancements in diagnostic, surgical, radiotherapy, and molecular therapy techniques, while its 5-year survival rate remains low. Molecular biomarkers provide prognostic value. Oxidative stress factors, such as reactive nitrogen species and ROS, are crucial in various stages of tumor progression, influencing cell transformation, proliferation, angiogenesis, and metastasis. ROS demonstrate dual roles, affecting tumor cells, hypoxia sensitivity, and the microenvironment. Comprehensive analysis of oxidative stress in LUAD has not been conducted to date. Therefore, we systematically investigated the regulatory patterns of oxidative stress in LUAD based on oxidative stress-related genes and correlated these patterns with cellular infiltration characteristics of the tumor immune microenvironment. The model utilizes single-factor Cox analysis to screen key differential genes with prognostic value and employs least absolute shrinkage and selection operator (LASSO) penalized Cox regression analysis to construct a prognostic-related prediction model. Ten candidate genes were selected based on this model. The risk score was constructed using the coefficients and expression levels of these ten genes. Furthermore, the impact of this risk score on overall survival (OS) was determined. Two genes with the most significant differential expression, SFTPB and S100P, were selected through qRT-PCR. Cell experiments including CCK-8, Edu, transwell assays confirmed their effects on lung cancer cells growth, consistent with the results of bioinformatics analysis. These findings suggested that this model held potential clinical value for evaluating the prognosis of lung adenocarcinoma.

Endometrial Cancer: A Pilot Study of the Tissue Microbiota.

The endometrium remains a difficult tissue for the analysis of microbiota, mainly due to the low bacterial presence and the sampling procedures. Among its pathologies, endometrial cancer has not yet been completely investigated for its relationship with microbiota composition. In this work, we report on possible correlations between endometrial microbiota dysbiosis and endometrial cancer. Women with endometrial cancer at various stages of tumor progression were enrolled together with women with a benign polymyomatous uterus as the control. Analyses were performed using biopsies collected at two specific endometrial sites during the surgery. This study adopted two approaches: the absolute quantification of the bacterial load, using droplet digital PCR (ddPCR), and the analysis of the bacterial composition, using a deep metabarcoding NGS procedure. ddPCR provided the first-ever assessment of the absolute quantification of bacterial DNA in the endometrium, confirming a generally low microbial abundance. Metabarcoding analysis revealed a different microbiota distribution in the two endometrial sites, regardless of pathology, accompanied by an overall higher prevalence of pathogenic bacterial genera in cancerous tissues. These results pave the way for future studies aimed at identifying potential biomarkers and gaining a deeper understanding of the role of bacteria associated with tumors.

Changes in subset distribution and impaired function of circulating natural killer cells in patients with colorectal cancer

Natural killer (NK) cells are closely associated with malignant tumor progression and metastasis. However, studies on their relevance in colorectal cancer (CRC) are limited. We aimed to comprehensively analyze the absolute counts, phenotypes, and function of circulating NK cells in patients with CRC using multiparametric flow cytometry. The distribution of NK cell subsets in the peripheral circulation of patients with CRC was significantly altered relative to the control group. This is shown by the decreased frequency and absolute count of CD56dimCD16+ NK cells with antitumor effects, contrary to the increased frequency of CD56bright NK and CD56dimCD16- NK cells with poor or ineffective antitumor effects. NK cells in patients with CRC were functionally impaired, with decreased intracellular interferon (IFN)-γ secretion and a significantly lower percentage of cell surface granzyme B and perforin expression. In addition, IFN-γ expression decreased significantly with the tumor stage progression. Based on a comprehensive analysis of the absolute counts, phenotypes, and functional markers of NK cells, we found an altered subset distribution and impaired function of circulating NK cells in patients with CRC.

CDKN2A copy number alteration in bladder cancer: Integrative analysis in patient-derived xenografts and cancer patients

Bladder cancer (BlCa) is an extensively heterogeneous disease that leads to great variability in tumor evolution scenarios and lifelong patients’ surveillance, emphasizing the need for modern minimally-invasive precision medicine. Herein, we have explored the clinical significance of copy-number alterations (CNAs) in BlCa. CNAs profiling was performed in 15 patient-derived xenografts (PDXs) and validated in TCGA-BLCA (n=408) and Lindgren et al. (n=143) cohorts. CDKN2A copy-number loss was identified as the most frequent CNA in bladder tumors, associated with reduced CDKN2A expression, tumors of papillary phenotype and prolonged PDX survival. Study’s screening cohort consisted of 243 BlCa patients and CDKN2A copy-number was assessed in genomic DNA and cell-free DNA (cfDNA) from 217 tumors and 189 pre-treatment serum samples, respectively. CDKN2A copy-number loss was correlated with superior disease-free and progression-free survival of non-muscle-invasive bladder cancer (NMIBC) patients. Moreover, higher CDKN2A index (CDKN2A/LEP ratio) in pre-treatment cfDNA was associated with advanced tumor stage, grade and short-term NMIBC progression to invasive disease, while multivariate models fitted for CDKN2A index in pre-treatment cfDNA offered superior risk-stratification of T1/high grade and EORTC high-risk patients, enhancing prediction of treatment outcome. CDKN2A copy-number status could serve as minimally-invasive tool in improving risk-stratification and supporting personalized prognosis in BlCa.

Overcoming Nutrient Stress: Integrin αvβ3-Driven Metabolic Adaptation Supports Tumor Initiation.

Nutrient stress accompanies several stages of tumor progression, including metastasis formation. Metabolic reprogramming is a hallmark of cancer, and it has been associated with stress tolerance and anchorage-independent cell survival. Adaptive responses are required to support cancer cell survival under these conditions. In this issue of Cancer Research, Nam and colleagues showed that the extracellular matrix (ECM) receptor integrin β3 was upregulated in lung cancer cells in response to nutrient starvation, resulting in increased cell survival that was independent from ECM binding. Delving into the molecular mechanisms responsible for this, the authors found that integrin β3 promoted glutamine metabolism and oxidative phosphorylation (OXPHOS) by activating a Src/AMPK/PGC1α signaling pathway. Importantly, in vivo experiments confirmed that OXPHOS inhibition suppressed tumor initiation in an orthotopic model of lung cancer, while β3 knockout completely abrogated tumor initiation. These observations indicate that targeting signaling pathways downstream of αvβ3 could represent a promising therapeutic avenue to prevent lung cancer progression and metastasis. See related article by Nam et al., p. 1630.

The comprehensive roles of lncRNA FAM99A/FAM99B in hepatocellular carcinoma: Expressions, regulatory mechanisms and functional pathway analysis

AimsThe incidence and mortality of liver hepatocellular carcinoma (LIHC) were increasing year by year. The aim of this study was to investigate the comprehensive roles of lncRNA FAM99A and FAM99B in LIHC. Main methodsAccording to the data of TCGA and GTEx, the expression levels of FAM99A and FAM99B in LIHC were evaluated, and the overall survival (OS), disease-free survival (DFS), immune cell infiltration and tumor stage were analyzed. The subcellular localization of FAM99A and FAM99B in various cancer cell lines was predicted by lncATLAS database. In addition, we also used ENCORI, KEGG, LinkedOmics, Metascape and other databases. It was verified by in vivo and in vitro experiments. Key findingsCompared with adjacent normal tissues, FAM99A and FAM99B were down-regulated in LIHC tissues, and significantly correlated with immune cell infiltration. With the progression of tumor stage and grade, the expression of FAM99A and FAM99B showed a decreasing trend, and the prognosis of patients were also poor. In addition, the biological functions, signaling pathways and protein interactions of FAM99A and FAM99B in LIHC were enriched to study the potential molecular mechanisms. The overlapping RNA binding proteins (RBP) of FAM99A and FAM99B mainly included CSTF2T, BCCIP, RBFOX2 and SF3B4. Finally, experiments showed that overexpression of FAM99A attenuated the proliferation, invasion, colony formation and tumor growth of LIHC cells. SignificanceTaken together, the above studies demonstrated that FAM99A and FAM99B had an inhibitory effect on the progression of LIHC, which might be promising diagnostic biomarkers and therapeutic targets for LIHC patients.

Understanding the Role of Connexins in Hepatocellular Carcinoma: Molecular and Prognostic Implications.

Connexins, a family of tetraspan membrane proteins forming intercellular channels localized in gap junctions, play a pivotal role at the different stages of tumor progression presenting both pro- and anti-tumorigenic effects. Considering the potential role of connexins as tumor suppressors through multiple channel-independent mechanisms, their loss of expression may be associated with tumorigenic activity, while it is hypothesized that connexins favor the clonal expansion of tumor cells and promote cell migration, invasion, and proliferation, affecting metastasis and chemoresistance in some cases. Hepatocellular carcinoma (HCC), characterized by unfavorable prognosis and limited responsiveness to current therapeutic strategies, has been linked to gap junction proteins as tumorigenic factors with prognostic value. Notably, several members of connexins have emerged as promising markers for assessing the progression and aggressiveness of HCC, as well as the chemosensitivity and radiosensitivity of hepatocellular tumor cells. Our review sheds light on the multifaceted role of connexins in HCC pathogenesis, offering valuable insights on recent advances in determining their prognostic and therapeutic potential.

Convolutional Neural Network Approach for White Blood Cells Classification

White blood cells (WBCs) or leukocytes are immune cells responsible for protecting the body against infections, engulfing foreign invaders, repairing tissue injuries, and resisting pathogens or diseases. WBCs such as macrophages, neutrophils, T cells, and natural killer cells (NK cells) are essential players in many important diseases. For example, in cancer, WBCs remodel the tumor extracellular matrix, facilitate tumor cell migration and invasion, and modulate tumor angiogenesis. Through these activities, WBCs promote all stages of tumor progression. The system is developed the DL algorithms such as convolutional neural network for predicting the types of blood cell disease. Finally, the system can estimate some performance metrics such as accuracy and error rate. Keywords—Image Segmentation, White blood cells, Convolutional Neural Network, Deep learning, Blood leukocytes segmentation.

Abstract 6208: Characterization of molecular evolution in myelodysplastic neoplasms

Abstract Myelodysplastic neoplasms (MDS) are hematopoietic stem cell disorders. The IPSS-Mol model, a scoring system that combines molecular and clinical features to stratify MDS patients, can identify individuals at high-risk of developing Acute Myeloid Leukemia (AML), enabling the targeted application of disease-modifying therapies. However, the molecular evolutionary patterns in MDS and their impact on Overall Survival (OS) and Leukemia-Free Survival (LFS) remain unexplored. Shifting from conventional static mutational analyses used by IPSS-Mol to a dynamic evolutionary approach, we employed the ASCETIC framework to analyze data from the IPSS-Mol Database (3,323 patients, 152 genes) and understand the dynamics of MDS progression. According to ASCETIC, genes related to DNA methylation displayed low ranks, indicating their tendency to manifest early in the progression of the disease. In contrast, genes associated with cellular signaling typically appeared in the later stages of tumor progression, while genes involved in RNA splicing exhibited intermediate ranks.Regarding molecular evolution in MDS, ASCETIC identified five clusters, each characterized by distinct evolutionary signatures and significantly different OS and LFS rates (p < 0.001). The median OS for the C1, C2, C3, C4, and C5 clusters were 79.3, 64.1, 28.9, 16.2, and 14.8 months, respectively. Subsequently, we compared ASCETIC's clusters with the different risk groups defined by the IPSS-Mol score. Among the 1158 patients initially classified as V-Low/M-Low risk by IPSS-Mol, ASCETIC identified 55 patients placed in C4 and C5. These patients exhibited lower OS (p = 0.005) and LFS (p = 0.02). Conversely, among the 905 IPSS-Mol M-High/V-High risk patients, ASCETIC identified 468 patients (51.7%) classified as low risk (C1-C2-C3), who were associated with longer median OS and LFS (p < 0.001). The differences between conventional mutational analyses and our evolutionary approach primarily, but not exclusively, arise from the evaluation of TP53 mutations and ASXL1. In the IPSS-Mol model, a higher risk is conferred if multi-hit TP53 alterations occur or in the presence of ASXL1 mutations.Conversely, ASCETIC assesses the impact of mutations differently. It considers not only the Variant Allele Frequencies of individual- and/or co-mutations but also the evolutionary trajectories that lead to TP53 and/or ASXL1. This approach results in different risk weights assigned to each evolutionary trajectory.Lastly, we identified patients with NPM1 mutations, classified as MDS patients in the original dataset but now categorized as AML according to 2022-WHO. Within this high-risk context, ASCETIC differentiated between patients with shorter and longer Overall Survival (OS) (p < 0.001).Our study reveals the influence of evolution on MDS prognosis, offering insights into the integration of the ASCETIC framework with the IPSS-Mol. Validation on external cohort is ongoing. Citation Format: Ivan Civettini, Valentina Crippa, Federica Malighetti, Matteo Villa, Andrea Aroldi, Fabrizio Cavalca, Alex Graudenzi, Lorenza Maria Borin, Luca Mologni, Rocco Piazza, Carlo Gambacorti-Passerini, Daniele Ramazzotti. Characterization of molecular evolution in myelodysplastic neoplasms [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 6208.

Abstract 287: Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of colorectal cancer

Abstract A new subset of cancer associated fibroblasts (CAFs) called antigen presenting CAF (apCAF) was recently identified, and modulation of the tumor immunity might be associated with tumor progression. We examined the markers of apCAF, such as IRF5, HLA-DRA and HLA-DQA1 by bioinformatics analyses of human colorectal cancer (CRC) datasets. The expression of apCAF markers were upregulated in CAFs isolated from human CRCs and liver metastases compared with their normal counterparts. Kaplan-Meir analysis of 172 human CRC samples clarified that the patients with apCAF marker positive expression in the tumor stroma had worse prognosis than that with negative expression. In addition, the origin of apCAFs was reported to be mesothelial cells, therefore, we established cancer-associated mesothelial cells (CAmes) from ascites of human CRC patients with peritoneal dissemination. Consistently, the expression of antigen-presentation markers was highly upregulated in CAmes, however, allo-mixed lymphocyte reaction analysis demonstrated that their stimulating activity of CD4+ T lymphocytes was diminished. In an in vivo study, we applied a rectal orthotopic transplant model with mouse tumor organoid (MTO) to investigate the effect of apCAF on tumor progressions. Using fluorescence-activated cell sorting (FACS), we examined the ratios of apCAF, myCAF, and iCAF in orthotopic rectal tumors. The percentage of apCAF was highest in the 6-week-tumors and the percentage of myCAF was highest in the 9-week-tumors. Interestingly, the percentage of apCAF was higher in liver metastases than in primary tumor, consistent with bioinformatics analysis. The apCAF increased during the stage of tumor growth, and the percentage of myCAF increase in the later stage of tumor progression with fibrosis. In summary, apCAF might be associated with tumor progression in CRC. Citation Format: Yasuhiro Fukui, Hiroaki Kasashima, Zizhou Wang, Ken Yonemitsu, Kishu Kitayama, Yuichiro Miki, Mami Yoshii, Tatsunari Fukuoka, Tatsuro Tamura, Masatsune Shibutani, Takahiro Toyokawa, Hiroaki Tanaka, Shigeru Lee, Masakazu Yashiro, Kiyoshi Maeda. Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of colorectal 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 287.

Abstract 86: Deep immuno-profiling of syngeneic tumor mouse models for preclinical studies

Abstract The identification of the major cellular players involved in the progression of a type of cancer is a key step for the success of new immunotherapies for personalized medicine. Immune cells play critical functions in cancer, and mice with intact immune systems are vital to understanding tumor immunology. It is however a daunting challenge as complex relationships interplay between tumor cells and the immune system. Each component of innate immunity or adaptive immunity, such as T lymphocytes, macrophages or neutrophils, may be directed to a pro- or anti-tumor function. In order to cope with the complexity of the tumor microenvironment, it is necessary to use an experimental approach capable of characterizing the heterogeneity of the cell types present in the tumor, the evolution of their relative proportion and their fine-tuned functional specificity. This approach leads to the identification of new cellular biomarkers of different stages of tumor progression in order to identify new targets for therapeutic time-window of and improve cancer diagnosis To decipher the impact of immunotherapy treatments involved in the anti-tumor response, cellular phenotyping of leukocytes infiltrating a tumor but also those present in peripheral organs is necessary. Essentially based on extracellular labeling, this primary screen aims to quantify the different cell populations present in a syngenic tumor models on B6/N or BalbC genetical backgrounds. In order to increase our understanding in the precise mode of action of anti-PD1 treatment at the cellular level in sensitive and unsensitive models, we investigated immunophenotypes and responses to immune checkpoint inhibitor (ICI) of several hallmark syngeneic tumor models (MC38, CT26, B16F10, B16-OVA, RENCA, EMT6) in immunocompetent mouse models by flow and mass cytometry. We compared growth kinetics and profiled the immune cell composition of tumor microenvironment (TME), draining lymph node (dLN) and blood in order to establish immune-phenotypic cell signatures that correlates with treatment efficacy. Supervised, unsupervised and integrative data analysis as well as visualization tools are used to identify significant changes across different experimental settings. Our results indicate that each model possesses a unique tumor-immune infiltrate profile that can be modulated with immunotherapies. Overall, these studies provide an important resource of highly-characterized syngeneic tumor model and highlight the importance of tumor immune landscape variance across models that will drive selecting the most appropriate model to test novel immunotherapeutic agents and enhance our translation of knowledge from syngeneic models to human tumors. Citation Format: Anais Joachim, Emilie Maturin, Marielle Mello, Lilia Hadjem, Magali Grange, Olivier Deas, Ana Zarubica, Bernard Malissen, Hervé Luche, François Romagne, Stéphanie Blanchin. Deep immuno-profiling of syngeneic tumor mouse models for preclinical studies [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 86.

Abstract 6812: Durable control of brain tumors by glioma inhibitory macrophages and IL33

Abstract Glioblastoma is the most common and deadly form of brain cancer. Even with aggressive treatment including surgery, chemotherapy, and radiotherapy, survival outcomes for newly diagnosed glioblastoma patients remains less than two years. Novel high throughput omics technologies have expanded our understanding of the role of innate immune system in brain tumors, that are generally believed to drive glioma progression and enable evasion of the adaptive immune system. However, targeting of this axis in the clinic remains an unmet opportunity. Previously, we discovered that the dual-function (secreted and nuclear) cytokine IL-33 is a crucial regulator of the inflammatory microenvironment that promotes glioma tumorigenesis through phenotypic and functional changes in the innate immune cell repertoire. Strikingly, when IL-33 is prevented from entering the nucleus, by deletion of its nuclear localization sequence (ΔNLS IL-33), but is still secreted, in vivo tumor growth is dramatically inhibited resulting in prolonged long-term survival. Using multiplex immunohistochemistry and spatial transcriptomics with temporal resolution across different stages of tumor progression, we identified a population of glioma-inhibitory macrophages (GIMs) unique to this suppressive environment. Assessment of GIMs in xenografts generated from patient brain tumor initiating cells found an enriched presence of these cells in xenografts with long-term survival (greater than 300 days) versus short-term survival (less than 100 days). The ability of GIMs to inhibit glioma progression was demonstrated when tumors established using a combination of ΔNLS IL-33 expressing cancer cells together with highly tumorigenic cells resulted in a growth inhibitory environment that significantly extended survival. A deeper molecular characterization of this phenotype and development of clinical strategies are currently underway. Citation Format: Shyam V. Menon, Xueqing Lun, Peipei Zeng, Jianbo Zhang, Bo Young Ahn, Henry Yu, Alisha Poole, Ngoc Ha Dang, Katalin Osz, Jennifer A. Chan, Daniela F. Quail, Stephen M. Robbins, Donna L. Senger. Durable control of brain tumors by glioma inhibitory macrophages and IL33 [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 6812.

Abstract 6926: Tracing clonal evolution and immune evasion in glioblastoma progression

Abstract Glioblastoma continues to be a daunting obstacle in oncology research, with its early-stage progression being particularly enigmatic. In a recent published work, we traced the clonal dynamics of glioblastoma evolution by the simultaneous transfer of PDGFB and genetic barcodes into mouse brains. We observed a continuous clonal loss during the acquisition of a malignant phenotype, underlined by the modulation of the levels of c-Myc expression and their functional targets. In this study, we delve deeper into the evolution of glioblastoma by transplanting multiclonal, early-stage glioma cells into multiple immunodeficient NOD-SCID mice. This experimental design allowed us to track the clonal dynamics over serial transplants, revealing how early-stage glioma clones acquire immune-evasive features capable of initiating tertiary tumors in immune-competent hosts. Through barcode sequencing and single-cell RNA sequencing of early-stage gliomas, coupled with bulk RNA sequencing of secondary and tertiary gliomas, we dissected the clonal and transcriptomic landscape of these tumors. We observed that, of the various clones composing primary tumors, just a few of them are predominating in subsequent passages. Furthermore, the clonal composition of secondary tumors derived from the same primary glioma showed partial overlap, indicating a partial predetermination in the development of immune-evasive behavior. Our intra- and inter-clonal transcriptomic analysis across various stages of tumor progression is shedding light on how new functional traits can emerge in gliomas. Further analysis of our data will determine whether it is the result of the expansion of clones already possessing these traits, or if they stem from functional adaptations within the clones themselves. Our insights further reinforce the concept of clonal competition, highlighting the significant influence of immune-system interactions in driving these competitive dynamics. Citation Format: Davide Ceresa, Sebastian A. Garcia Mena, Francesca Piaggio, Appolloni Irene, Daniela Marubbi, Paolo Malatesta. Tracing clonal evolution and immune evasion in glioblastoma progression [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 6926.

Abstract 1566: Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease which is thought to be initiated by colon cancer stem cells according to cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcome. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and CSC-associated invasive phenotype based on patient-derived 3D cell culture models (PD3D), patient-derived xenografts (PDX) and human CRC cell lines. We showed that CD44-enriched CSCs from PD3D models express significantly higher levels of MACC1 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PD3D and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with CSC markers CD44, NANOG and LGR5 in PD3D models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter uncovering the long-known CSC marker LGR5 as novel essential signaling mediator used by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by significant positive correlation of MACC1 and LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis-inducer MACC1 act as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs, prevent cancer recurrence and distant metastasis formation. Citation Format: Müge Erdem, Kyung Hwan Lee, Markus Hardt, Joseph Regan, Dennis Kobelt, Wolfgang Walther, Christian Regenbrecht, Ulrike S. Stein. Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal 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 1566.

Abstract 441: Bioluminescent dehydrogenase assays: A novel approach for metabolic profiling and inhibitor screening in cancer biology

Abstract Dehydrogenases play a pivotal role in cellular metabolism, acting as gatekeepers in key metabolic pathways. Their activity reflects the metabolic state of the cell, which is particularly relevant in the context of cancer and the immune response, where metabolic reprogramming is a hallmark of tumor progression and differentiation stages. Here, we present the development and application of novel bioluminescent dehydrogenase assays for direct measurement of metabolic activity in cell lysates. Our assays are highly sensitive, requiring as few as 100-500 cells, and have been successfully miniaturized to a 384-well plate format conducive to high-throughput screening. This technological advance allows for the precise monitoring of dehydrogenase activity across the pentose phosphate pathway, glycolysis, and the mitochondrial tricarboxylic acid (TCA) cycle. We demonstrate how shifts in dehydrogenase activity can serve as indicators of the metabolic status of cells, and their modulation during differentiation. Moreover, our bioluminescent assays provide a rapid screening platform for pathway-specific metabolic inhibitors, with potential applications in identifying new therapeutic targets within cancer metabolism. Our data reveal a dynamic landscape of metabolic activity during T cell maturation, underscoring the potential of dehydrogenase activity profiling in cancer immunobiology and the development of targeted cancer therapies. Citation Format: Kim Haupt, Natasha Karassina, Anthony Lauer, Kayla Sylvester, Jolanta Vidugiriene. Bioluminescent dehydrogenase assays: A novel approach for metabolic profiling and inhibitor screening in cancer biology [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 441.