Subpopulation Of Cancer Cells Research Articles

Induction of Multiple Alternative Mitogenic Signaling Pathways Accompanies the Emergence of Drug-Tolerant Cancer Cells.

Drug resistance can evolve from a subpopulation of cancer cells that initially survive drug treatment and then gradually form a pool of drug-tolerant cells. Several studies have pinpointed the activation of a specific bypass pathway that appears to provide the critical therapeutic target for preventing drug tolerance. Here, we take a systems-biology approach, using proteomics and genomics to examine the development of drug tolerance to EGFR inhibitors in EGFR-mutant lung adenocarcinoma cells and BRAF inhibitors in BRAF-mutant melanoma cells. We found that there are numerous alternative mitogenic pathways that become activated in both cases, including YAP, STAT3, IGFR1, and phospholipase C (PLC)/protein kinase C (PKC) pathways. Our results suggest that an effective therapeutic strategy to prevent drug tolerance will need to take multiple alternative mitogenic pathways into account rather than focusing on one specific pathway.

Tropomyosin1 isoforms underlie epithelial to mesenchymal plasticity, metastatic dissemination, and resistance to chemotherapy in high-grade serous ovarian cancer

Phenotypic plasticity, defined as the ability of individual cells with stable genotypes to exert different phenotypes upon exposure to specific environmental cues, represent the quintessential hallmark of the cancer cell en route from the primary lesion to distant organ sites where metastatic colonization will occur. Phenotypic plasticity is driven by a broad spectrum of epigenetic mechanisms that allow for the reversibility of epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions (EMT/MET). By taking advantage of the co-existence of epithelial and quasi-mesenchymal cells within immortalized cancer cell lines, we have analyzed the role of EMT-related gene isoforms in the regulation of epithelial mesenchymal plasticity (EMP) in high grade serous ovarian cancer. When compared with colon cancer, a distinct spectrum of downstream targets characterizes quasi-mesenchymal ovarian cancer cells, likely to reflect the different modalities of metastasis formation between these two types of malignancy, i.e. hematogenous in colon and transcoelomic in ovarian cancer. Moreover, upstream RNA-binding proteins differentially expressed between epithelial and quasi-mesenchymal subpopulations of ovarian cancer cells were identified that underlie differential regulation of EMT-related isoforms. In particular, the up- and down-regulation of RBM24 and ESRP1, respectively, represent a main regulator of EMT in ovarian cancer cells. To validate the functional and clinical relevance of our approach, we selected and functionally analyzed the Tropomyosin 1 gene (TPM1), encoding for a protein that specifies the functional characteristics of individual actin filaments in contractile cells, among the ovarian-specific downstream AS targets. The low-molecular weight Tpm1.8/9 isoforms are specifically expressed in patient-derived ascites and promote invasion through activation of EMT and Wnt signaling, together with a broad spectrum of inflammation-related pathways. Moreover, Tpm1.8/9 expression confers resistance to taxane- and platinum-based chemotherapy. Small molecule inhibitors that target the Tpm1 isoforms support targeting Tpm1.8/9 as therapeutic targets for the development of future tailor-made clinical interventions.

Long Non-Coding RNAs as Regulators for Targeting Breast Cancer Stem Cells and Tumor Immune Microenvironment: Biological Properties and Therapeutic Potential.

Breast cancer stem cells (BCSCs) is a subpopulation of cancer cells with self-renewal and differentiation capacity, have been suggested to give rise to tumor heterogeneity and biologically aggressive behavior. Accumulating evidence has shown that BCSCs play a fundamental role in tumorigenesis, progression, and recurrence. The development of immunotherapy, primarily represented by programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors, has greatly changed the treatment landscape of multiple malignancies. Recent studies have identified pervasive negative associations between cancer stemness and anticancer immunity. Stemness seems to play a causative role in the formation of cold tumor immune microenvironment (TIME). The multiple functions of long non-coding RNAs (lncRNAs) in regulating stemness and immune responses has been recently highlighted in breast cancer. The review focus on lncRNAs and keys pathways involved in the regulation of BCSCs and TIME. Potential clinical applications using lncRNAs as biomarkers or therapies will be discussed.

P66ShcA promotes malignant breast cancer phenotypes by alleviating energetic and oxidative stress

Significant efforts have focused on identifying targetable genetic drivers that support the growth of solid tumors and/or increase metastatic ability. During tumor development and progression to metastatic disease, physiological and pharmacological selective pressures influence parallel adaptive strategies within cancer cell sub-populations. Such adaptations allow cancer cells to withstand these stressful microenvironments. This Darwinian model of stress adaptation often prevents durable clinical responses and influences the emergence of aggressive cancers with increased metastatic fitness. However, the mechanisms contributing to such adaptive stress responses are poorly understood. We now demonstrate that the p66ShcA redox protein, itself a ROS inducer, is essential for survival in response to physiological stressors, including anchorage independence and nutrient deprivation, in the context of poor outcome breast cancers. Mechanistically, we show that p66ShcA promotes both glucose and glutamine metabolic reprogramming in breast cancer cells, to increase their capacity to engage catabolic metabolism and support glutathione synthesis. In doing so, chronic p66ShcA exposure contributes to adaptive stress responses, providing breast cancer cells with sufficient ATP and redox balance needed to withstand such transient stressed states. Our studies demonstrate that p66ShcA functionally contributes to the maintenance of aggressive phenotypes and the emergence of metastatic disease by forcing breast tumors to adapt to chronic and moderately elevated levels of oxidative stress.

Bioengineered hydrogels enhance ex vivo preservation of patient-derived tumor explants for drug evaluation

Ex vivo patient-derived tumor slices (PDTS) are currently limited by short-term viability in culture. Here, we show how bioengineered hydrogels enable the identification of key matrix parameters that significantly enhance PDTS viability compared to conventional culture systems. As demonstrated using single-cell RNA sequencing and high-dimensional flow cytometry, hydrogel-embedded PDTS tightly preserved cancer, cancer-associated fibroblast, and various immune cell populations and subpopulations in the corresponding original tumor. Cell-cell communication networks within the tumor microenvironment, including immune checkpoint ligand-receptor interactions, were also maintained. Remarkably, our results from a co-clinical trial suggest hydrogel-embedded PDTS may predict sensitivity to immune checkpoint inhibitors (ICIs) in head and neck cancer patients. Further, we show how these longer term-cultured tumor explants uniquely enable the sampling and detection of temporal evolution in molecular readouts when treated with ICIs. By preserving the compositional heterogeneity and complexity of patient tumors, hydrogel-embedded PDTS provide a valuable tool to facilitate experiments targeting the tumor microenvironment.

Dielectrophoretic enrichment of live chemo-resistant circulating-like pancreatic cancer cells from media of drug-treated adherent cultures of solid tumors.

Due to low numbers of circulating tumor cells (CTCs) in liquid biopsies, there is much interest in enrichment of alternative circulating-like mesenchymal cancer cell subpopulations from in vitro tumor cultures for utilization within molecular profiling and drug screening. Viable cancer cells that are released into the media of drug-treated adherent cancer cell cultures exhibit anoikis resistance or anchorage-independent survival away from their extracellular matrix with nutrient sources and waste sinks, which serves as a pre-requisite for metastasis. The enrichment of these cell subpopulations from tumor cultures can potentially serve as an in vitro source of circulating-like cancer cells with greater potential for scale-up in comparison with CTCs. However, these live circulating-like cancer cell subpopulations exhibit size overlaps with necrotic and apoptotic cells in the culture media, which makes it challenging to selectively enrich them, while maintaining them in their suspended state. We present optimization of a flowthrough high frequency (1 MHz) positive dielectrophoresis (pDEP) device with sequential 3D field non-uniformities that enables enrichment of the live chemo-resistant circulating cancer cell subpopulation from an in vitro culture of metastatic patient-derived pancreatic tumor cells. Central to this strategy is the utilization of single-cell impedance cytometry with gates set by supervised machine learning, to optimize the frequency for pDEP, so that live circulating cells are selected based on multiple biophysical metrics, including membrane physiology, cytoplasmic conductivity and cell size, which is not possible using deterministic lateral displacement that is solely based on cell size. Using typical drug-treated samples with low levels of live circulating cells (<3%), we present pDEP enrichment of the target subpopulation to ∼44% levels within 20 minutes, while rejecting >90% of dead cells. This strategy of utilizing single-cell impedance cytometry to guide the optimization of dielectrophoresis has implications for other complex biological samples.

EXOSC5 maintains cancer stem cell activity in endometrial cancer by regulating the NTN4/integrin β1 signalling axis.

Endometrial carcinoma (EC) is a common type of uterine cancer in developed countries, originating from the uterine epithelium. The incidence rate of EC in Taiwan has doubled from 2005. Cancer stem cells (CSCs) are a subpopulation of cancer cells that have high tumorigenicity and play a crucial role in the malignant processes of cancer. Targeting molecules associated with CSCs is essential for effective cancer treatments. This study delves into the role of Exosome component 5 (EXOSC5) in EC. Data from The Cancer Genome Atlas suggests a correlation between high EXOSC5 mRNA expression and unfavorable EC prognosis. EXOSC5 knockdown diminished EC-CSC self-renewal and reduced expression of key cancer stemness proteins, including c-MYC and SOX2. Intriguingly, this knockdown significantly curtailed tumorigenicity and CSC frequency in EC tumor spheres. A mechanistic examination revealed a reduction in netrin4 (NTN4) levels in EXOSC5-depleted EC cells. Moreover, NTN4 treatment amplified EC cell CSC activity and, when secreted, NTN4 partnered with integrin β1, subsequently triggering the FAK/SRC axis to elevate c-MYC activity. A clear positive relation between EXOSC5 and NTN4 was evident in 93 EC tissues. In conclusion, EXOSC5 augments NTN4 expression, activating c-MYC via the integrin β1/FAK/SRC pathway, offering potential avenues for EC diagnosis and treatment.

TET1 regulates stem cell properties and cell cycle of Cancer stem cells in triple-negative breast cancer via DNA demethylation

The role of cancer stem cells in metastasis, recurrence, and resistance to conventional therapies is significant. Addressing these cells could potentially decrease cancer reoccurrences and mortality rates. TET1, a crucial gene involved in stem cell self-renewal and potency, may also play a part in cancer stem cells, which warrants further research.To explore the role of TET1 in cancer stem cells, we conducted experiments involving loss and gain. We then analyzed factors such as migration, invasion, cell cycle, cell viability, mammosphere formation, and the CD44+/CD24- subpopulation of cancer cells. We also investigate the influence of TET1 on CCNB1, CDK1, and OCT4.Our study reveals that TET1 can regulate the phenotype of cancer stem cells via OCT4. Additionally, it can control the cell cycle by increasing CDK1 and CCNB1 levels. These findings suggest that targeting DNA methylation and TET1 could be an effective strategy to overcome obstacles posed by Cancer stem cells.Our research also indicates that TET1 can influence the phenotype of cancer stem cells and the cell cycle of breast cancer cells potentially through OCT4, CCNB1, and CDK1. This highlights the importance of TET1 in breast cancer cases and suggests a potential therapeutic approach through DNA methylation and modulation of TET1.

Mitochondrial Dynamics in Ovarian Cancer: Pathophysiology and Therapeutic Implications

Background: Ovarian cancer is often characterized by aggressive growth and chemoresistance, leading to a poor prognosis. The energy and nutrient acquisition through metabolic reprogramming has been reported to facilitate cancer cell proliferation, invasion, and metastasis. Therefore, a therapeutic strategy to consider is to rewire energy metabolism. Mitochondrial dynamics have a profound impact on the metabolic profiles. In this review, we summarize the current understanding of the molecular mechanisms governing mitochondrial dynamics and their impact on cell proliferation and invasion and discuss future perspectives for therapeutic strategies and research directions. Methods: A search was conducted for literature published up to 30 June 2023 using the online databases PubMed and Google Scholar in this narrative literature review. Results: Mitochondria are essential for regulating metabolic reprogramming to meet the increasing energy demand for rapid cancer cell proliferation and invasion. A metabolic switch from OXPHOS to glycolysis may promote invasion, and OXPHOS-driven metabolism may be associated with proliferation, chemoresistance, and stemness. Many ovarian cancer cells are known to favor glycolysis over OXPHOS, but the opposite takes place in the subpopulation of cancer cells. The preference for glycolysis versus OXPHOS in ovarian cancer cells may be determined by histopathologic types, the unique genetic profile of energy metabolism, and intrinsic (e.g., oncogenic signaling) and extrinsic (e.g., nutritional status and hypoxia) factors. Conclusions: Preclinical studies suggest that mitochondrial dynamics regulators have therapeutic potential in ovarian cancer, but some factors limit their beneficial effects.

Flow Sorting, Whole Genome Amplification and Next-Generation Sequencing as Combined Tools to Study Heterogeneous Acute Lymphoblastic Leukemia.

Next-generation sequencing (NGS) methods have been introduced for immunoglobulin (IG)/T-cell receptor (TR) gene rearrangement analysis in acute lymphoblastic leukemia (ALL) and lymphoma (LBL). These methods likely constitute faster and more sensitive approaches to analyze heterogenous cases of ALL/LBL, yet it is not known whether gene rearrangements constituting low percentages of the total sequence reads represent minor subpopulations of malignant cells or background IG/TR gene rearrangements in normal B-and T-cells. In a comparison of eight cases of B-cell precursor ALL (BCP-ALL) using both the EuroClonality NGS method and the IdentiClone multiplex-PCR/gene-scanning method, the NGS method identified between 29% and 139% more markers than the gene-scanning method, depending on whether the NGS data analysis used a threshold of 5% or 1%, respectively. As an alternative to using low thresholds, we show that IG/TR gene rearrangements in subpopulations of cancer cells can be discriminated from background IG/TR gene rearrangements in normal B-and T-cells through a combination of flow cytometry cell sorting and multiple displacement amplification (MDA)-based whole genome amplification (WGA) prior to the NGS. Using this approach to investigate the clonal evolution in a BCP-ALL patient with double relapse, clonal TR rearrangements were found in sorted leukemic cells at the time of second relapse that could be identified at the time of diagnosis, below 1% of the total sequence reads. These data emphasize that caution should be exerted when interpreting rare sequences in NGS experiments and show the advantage of employing the flow sorting of malignant cell populations in NGS clonality assessments.

Targeting hedgehog-driven mechanisms of drug-resistant cancers.

Due to the cellular plasticity that is inherent to cancer, the acquisition of resistance to therapy remains one of the biggest obstacles to patient care. In many patients, the surviving cancer cell subpopulation goes on to proliferate or metastasize, often as the result of dramatically altered cell signaling and transcriptional pathways. A notable example is the Hedgehog (Hh) signaling pathway, which is a driver of several cancer subtypes and aberrantly activated in a wide range of malignancies in response to therapy. This review will summarize the field's current understanding of the many roles played by Hh signaling in drug resistance and will include topics such as non-canonical activation of Gli proteins, amplification of genes which promote tolerance to chemotherapy, the use of hedgehog-targeted drugs and tool compounds, and remaining gaps in our knowledge of the transcriptional mechanisms at play.

Single-Cell Diagnosis of Cancer Drug Resistance through the Differential Endocytosis of Nanoparticles between Drug-Resistant and Drug-Sensitive Cancer Cells.

Single-cell diagnosis of cancer drug resistance is highly relevant for cancer treatment, as it can be used to identify the subpopulations of drug-resistant cancer cells, reveal the sensitivity of cancer cells to treatment, and monitor the progress of cancer drug resistance. However, simple and effective methods for cancer drug resistance detection at the single-cell level are still lacking in laboratory and clinical studies. Inspired by the fact that nanoparticles with diverse physicochemical properties would generate distinct and specific interactions with drug-resistant and drug-sensitive cancer cells, which have distinctive molecular signatures, here, we have synthesized a library of fluorescent nanoparticles with various sizes, surface charges, and compositions (SiO2 nanoparticles (SNPs), organic PS-co-PAA nanoparticles (ONPs), and ZIF-8 nanoparticles (ZNPs)), thus demonstrating that the composition has a critical influence on the interaction of nanoparticles with drug-resistant cancer cells. Furthermore, the clathrin/caveolae-independent endocytosis of ZNPs together with the P-glycoprotein-related decreased cell membrane fluidity resulted in a lower cellular accumulation of ZNPs in drug-resistant cancer cells, consequently causing the distinct cellular accumulation of ZNPs between the drug-resistant and drug-sensitive cancer cells. This difference was further quantified by detecting the fluorescence signals generated by the accumulation of nanoparticles at the single-cell level via flow cytometry. Our findings provide another insight into the nanoparticle-cell interactions and offer a promising platform for the diagnosis of cancer drug resistance of various cancer cells and clinical cancer samples at the single-cell level.

MSC.sensor: Capturing cancer cell interactions with stroma for functional profiling.

Mesenchymal stromal cells (MSCs) contribute to the microenvironment regulating normal and malignant hematopoiesis, and thus may support subpopulations of cancer cells to escape therapeutic pressure. Here, we engineered bone marrow MSCs to express a synthetic CD19-sensor receptor to detect and display interacting primary CD19+ leukemia cells in coculture. This implementation provides a versatile platform facilitating ex vivo drug response profiling of primary CD19+ leukemia cells in coculture with high-sensitivity and scalability.

High expression of DNMT3A and DNMT3B regulatory factors of TGFB in non-neoplastic liver tissues of HCC.

Increased expression of TGFB regulatory factors DNMT3A and DNMT3B in non-neoplastic liver tissues of HCC patients is the goal of this study. Furthermore, we demonstrate that TGF- is capable of elevating the percentage of CD133+ cells present in liver cancer cell lines in a manner that is both consistent and long-lasting over several cell divisions. This process is linked to stable alterations in DNA methylation that occur over the whole of the genome and continue even after cell division. In addition, the silencing of de novo DNA methyl-transferases with siRNA is able to inhibit the phenotypic changes that are induced by TGF-. According to the findings of our research, there is a self-sustaining interaction between the DNA methylation machinery and the TGF- signaling pathway, which may be significant in the development of cellular phenotypes. CD133 positive and negative fractions expand within liver cancer cell lines in proportions that remain stable throughout time. In contrast to their CD133- counterparts, MACS-sorted CD133+ Huh7cells demonstrated the ability to shape themselves into spheres when grown under non-attachment circumstances. This study also found that the TGF- is responsible for the de novo induction of CD133, which is linked to an increase in the expression of DNMT3 genes and there is a correlation between the TGF-induced transition in the cell subpopulation and a distinct DNA methylome. TGF- has the potential to generate genome-wide alterations in DNA methylation, which ultimately leads to a persistent shift in the fraction of liver cancer cell subpopulations.

Decoding Cancer Evolution: Integrating Genetic and Non-Genetic Insights.

The development of cancer begins with cells transitioning from their multicellular nature to a state akin to unicellular organisms. This shift leads to a breakdown in the crucial regulators inherent to multicellularity, resulting in the emergence of diverse cancer cell subpopulations that have enhanced adaptability. The presence of different cell subpopulations within a tumour, known as intratumoural heterogeneity (ITH), poses challenges for cancer treatment. In this review, we delve into the dynamics of the shift from multicellularity to unicellularity during cancer onset and progression. We highlight the role of genetic and non-genetic factors, as well as tumour microenvironment, in promoting ITH and cancer evolution. Additionally, we shed light on the latest advancements in omics technologies that allow for in-depth analysis of tumours at the single-cell level and their spatial organization within the tissue. Obtaining such detailed information is crucial for deepening our understanding of the diverse evolutionary paths of cancer, allowing for the development of effective therapies targeting the key drivers of cancer evolution.

Orai3 Calcium Channel Contributes to Oral/Oropharyngeal Cancer Stemness through the Elevation of ID1 Expression.

Emerging evidence indicates that intracellular calcium (Ca2+) levels and their regulatory proteins play essential roles in normal stem cell proliferation and differentiation. Cancer stem-like cells (CSCs) are subpopulations of cancer cells that retain characteristics similar to stem cells and play an essential role in cancer progression. Recent studies have reported that the Orai3 calcium channel plays an oncogenic role in human cancer. However, its role in CSCs remains underexplored. In this study, we explored the effects of Orai3 in the progression and stemness of oral/oropharyngeal squamous cell carcinoma (OSCC). During the course of OSCC progression, the expression of Orai3 exhibited a stepwise augmentation. Notably, Orai3 was highly enriched in CSC populations of OSCC. Ectopic Orai3 expression in non-tumorigenic immortalized oral epithelial cells increased the intracellular Ca2+ levels, acquiring malignant growth and CSC properties. Conversely, silencing of the endogenous Orai3 in OSCC cells suppressed the CSC phenotype, indicating a pivotal role of Orai3 in CSC regulation. Moreover, Orai3 markedly increased the expression of inhibitor of DNA binding 1 (ID1), a stemness transcription factor. Orai3 and ID1 exhibited elevated expression within CSCs compared to their non-CSC counterparts, implying the functional importance of the Orai3/ID1 axis in CSC regulation. Furthermore, suppression of ID1 abrogated the CSC phenotype in the cell with ectopic Orai3 overexpression and OSCC. Our study reveals that Orai3 is a novel functional CSC regulator in OSCC and further suggests that Orai3 plays an oncogenic role in OSCC by promoting cancer stemness via ID1 upregulation.

Phenotypically sorted highly and weakly migratory triple negative breast cancer cells exhibit migratory and metastatic commensalism

BackgroundIntratumor heterogeneity is a well-established hallmark of cancer that impedes cancer research, diagnosis, and treatment. Previously, we phenotypically sorted human breast cancer cells based on migratory potential. When injected into mice, highly migratory cells were weakly metastatic and weakly migratory cells were highly metastatic. The purpose of this study was to determine whether these weakly and highly migratory cells interact with each other in vitro or in vivo.MethodsTo assess the relationship between heterogeneity in cancer cell migration and metastatic fitness, MDA-MB-231 and SUM159PT triple negative breast cancer cells were phenotypically sorted into highly migratory and weakly migratory subpopulations and assayed separately and in a 1:1 mixture in vitro and in vivo for metastatic behaviors. Unpaired, two-tailed Student’s t-tests, Mann–Whitney tests, ordinary, one-way ANOVAs, and Kruskal–Wallis H tests were performed as appropriate with p < 0.05 as the cutoff for statistical significance.ResultsWhen highly and weakly migratory cells are co-seeded in mixed spheroids, the weakly migratory cells migrated farther than weakly migratory only spheroids. In mixed spheroids, leader–follower behavior occurred with highly migratory cells leading the weakly migratory cells in migration strands. When cell suspensions of highly migratory, weakly migratory, or a 1:1 mixture of both subpopulations were injected orthotopically into mice, both the mixed cell suspensions and weakly migratory cells showed significant distal metastasis, but the highly migratory cells did not metastasize significantly to any location. Notably, significantly more distal metastasis was observed in mice injected with the 1:1 mixture compared to either subpopulation alone.ConclusionsThis study suggests that weakly migratory cells interact with highly migratory cells in a commensal fashion resulting in increased migration and metastasis. Together, these findings indicate that cancer cell subpopulation migration ability does not correlate with metastatic potential and that cooperation between highly migratory and weakly migratory subpopulations can enhance overall metastatic fitness.

Targeting drug-tolerant cells: A promising strategy for overcoming acquired drug resistance in cancer cells.

Drug resistance remains the greatest challenge in improving outcomes for cancer patients who receive chemotherapy and targeted therapy. Surmounting evidence suggests that a subpopulation of cancer cells could escape intense selective drug treatment by entering a drug-tolerant state without genetic variations. These drug-tolerant cells (DTCs) are characterized with a slow proliferation rate and a reversible phenotype. They reside in the tumor region and may serve as a reservoir for resistant phenotypes. The survival of DTCs is regulated by epigenetic modifications, transcriptional regulation, mRNA translation remodeling, metabolic changes, antiapoptosis, interactions with the tumor microenvironment, and activation of signaling pathways. Thus, targeting the regulators of DTCs opens a new avenue for the treatment of therapy-resistant tumors. In this review, we first provide an overview of common characteristics of DTCs and the regulating networks in DTCs development. We also discuss the potential therapeutic opportunities to target DTCs. Last, we discuss the current challenges and prospects of the DTC-targeting approach to overcome acquired drug resistance. Reviewing the latest developments in DTC research could be essential in discovering of methods to eliminate DTCs, which may represent a novel therapeutic strategy for preventing drug resistance in the future.

Profiling single cancer cell metabolism via high-content SRS imaging with chemical sparsity.

Metabolic reprogramming in a subpopulation of cancer cells is a hallmark of tumor chemoresistance. However, single-cell metabolic profiling is difficult because of the lack of a method that can simultaneously detect multiple metabolites at the single-cell level. In this study, through hyperspectral stimulated Raman scattering (hSRS) imaging in the carbon-hydrogen (C-H) window and sparsity-driven hyperspectral image decomposition, we demonstrate a high-content hSRS (h2SRS) imaging approach that enables the simultaneous mapping of five major biomolecules, including proteins, carbohydrates, fatty acids, cholesterol, and nucleic acids at the single-cell level. h2SRS imaging of brain and pancreatic cancer cells under chemotherapy revealed acute and adapted chemotherapy-induced metabolic reprogramming and the unique metabolic features of chemoresistance. Our approach is expected to facilitate the discovery of therapeutic targets to combat chemoresistance. This study illustrates a high-content, label-free chemical imaging approach that measures metabolic profiles at the single-cell level and warrants further research on cellular metabolism.

Abstract PR005: An atlas of cellular heterogeneity in primary and metastatic renal cell carcinomas

Abstract Renal cell carcinoma (RCC) tumors harbor various layers of intra-tumor heterogeneity at the genetic, transcriptomic, and cellular levels. A series of recent studies have underlined the importance of charting this cellular variability to understand the cell of origin of various RCCs and the mechanisms that underlie their response to therapy. However, the regulatory factors driving intra-tumor heterogeneity in RCCs, the role of this heterogeneity in driving metastasis, and its association with clinical outcomes are mostly unknown. To address these questions, we have generated a cellular map of transcriptional heterogeneity in RCCs, which consists of &amp;gt;100,000 single-cell expression profiles from six patient-derived xenografts (PDX) models, including models derived from primary and brain metastatic tumors, as well as ten primary and six metastatic patient tumor tissues. We also developed a computational method for Gene Expression Decomposition and Integration (GEDI) that enables seamless integration of single-cell transcriptomic data across heterogeneous cancer samples while providing interpretable axes of variation. Using GEDI, we performed a fine-grained analysis of the heterogeneity of cancer cells and found that, despite patient-specific differences, common sources of intra-tumor heterogeneity exist across samples, driven by variable activity of pathways such as TNF-α/NF-κB signaling and oxidative phosphorylation (OxPhos). Analysis of data from our PDX models confirmed that this heterogeneity was stable and reproducible. A major source of intra-tumor heterogeneity was hypoxia signaling, even in VHL-deficient clear cell RCC samples (ccRCC), challenging the traditional view that ccRCC cells have a uniform pseudo-hypoxic status due to VHL inactivation. Regulatory network activity projection allowed us to disentangle the contributions of Hypoxia Inducible Factors HIF1A and HIF2A, revealing their divergent regulatory programs: while HIF1A is associated with cell-cycle and proliferation signatures, the activity of HIF2A correlates with epithelial-mesenchymal transition (EMT) within tumors, which we validated by RNA sequencing of HIF1A and EPAS1 knockdown in VHL-negative cells. Next, we used GEDI to study the cell state changes that occur during metastasis in both neoplastic and non-neoplastic tumor-infiltrating cells. By examining the differences in gene expression patterns between primary and metastatic samples, we identified various pathways that are transcriptionally activated during this transition, including EMT and Oxphos. We also uncovered distinct subpopulations of cancer cells with high gene expression changes associated with a metastatic profile. Analysis of the ligand-receptor interactions in the tumor microenvironment revealed that interactions from malignant cells to immune cells were over-represented in metastatic tumors. Overall, our cellular atlas has uncovered a complex continuum of cell states in RCCs, highlighting various drivers of the intra-tumor heterogeneity and establishing various cell states associated with metastasis. Citation Format: Ariel Madrigal, Minjun Kim, Adrien Osakwe, Tianyuan Lu, Zohreh Mehrjoo, Elham Moslemi, Rick Farouni, Larisa Morales-Soto, Yu Chang Wang, Matthew Dankner, Haig Djambazian, Kevin Petrecca, Jonathan Spicer, Fadi Brimo, Peter Siegel, Morag Park, Jiannis Ragoussis, Simon Tanguay, Yasser Riazalhosseini, Hamed S. Najafabadi. An atlas of cellular heterogeneity in primary and metastatic renal cell carcinomas [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR005.