Subtypes Of Cancer Cells Research Articles

Abstract 1221: Comprehensive cell-type classification of tumor and normal cells from single cell RNA sequencing in pan cancer settings

Abstract Single-cell RNA sequencing (scRNA-seq) allows for the study of the transcriptome at a cellular level, where populations of cells are annotated based on the expression of marker genes, providing a tool to gain cell-specific, subtle insights on cancer biology. However, precise annotation of cell type remains a challenge, hindering the efficiency of data interpretation. Several existing tools for cell-type annotation have been developed to improve resolution and reproducibility, yet their performance is reduced when the reference dataset contains many cell types, subclasses of similar cell types, or malignant cells. Interpatient malignant cell heterogeneity often leads to reduced accuracy when classifying cancer cells as most methods rely on correlations to a reference from a different source. Given the challenges in the annotation of scRNA-seq data of cancer and its high impact for elucidating mechanisms associated with tumor heterogeneity, pathogenesis, and treatment, we developed a comprehensive, hierarchically organized, multi-layered classifier spanning diverse malignant and normal cells of the tumor microenvironment. We found that performance improves when each layer focuses on a smaller number of classes and each cell sequentially moves down a series of classifiers with increased cell type resolution. When applied to an external validation dataset of over 300 primary solid tumor biopsies spanning diverse cancer types, the classifier accurately annotated the tissue of origin of malignant cells, and relevant subtypes of stromal and blood cells, with average F1 scores of 0.91, 0.95 and 0.99 respectively. Using confidence thresholds at each layer, the classifier abstains from classifying ambiguous cells. We applied 4 existing annotators provided with the same reference to the external test dataset and found that cancer cells are misclassified or unclassified, while the blood and stromal cells are accurately classified, highlighting our tool’s unique ability to classify cancer cells. Moreover, we applied our classifier’s to scRNA-seq data derived from breast cancer metastasis to the liver and were able to uncover the tissue of origin, demonstrating the potential use for determining the source of a metastatic tumour. Finally, given that our classifier is modular, we leveraged two recently published single cell breast cancer atlases to add a breast cancer subtype classification layer, that consistently identified the correct clinical subtype of single breast cancer cells in external data. This study provides a flexible model for the annotation of cells comprising the tumor microenvironment in pan cancer settings, while existing methods require tissue-specific references for every cancer type. Our classifier provides a powerful method for investigating intercellular communication pathways between tumor cells and non-malignant cells of the tumor microenvironment. Citation Format: Ido Nofech-Mozes, Philip Awadalla, Sagi Abelson. Comprehensive cell-type classification of tumor and normal cells from single cell RNA sequencing in pan cancer settings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1221.

CCR5 activation and endocytosis in circulating tumor-derived cells isolated from the blood of breast cancer patients provide information about clinical outcome

BackgroundCCR5 is a motility chemokine receptor implicated in tumor progression, whose activation and subsequent endocytosis may identify highly aggressive breast cancer cell subtypes likely to spread into the circulatory system.MethodsThe MDA-MB-231 cell line was used to model and visualize CCR5 activation by stimulation with RANTES, in an effort to quantify CCR5 endocytosis from the cell surface to the perinuclear space. CCR5 expression was then examined in tumor-associated cells (TACs), consisting of circulating tumor cells and circulating stromal cells, isolated from the peripheral blood of 54 metastatic breast cancer (mBC) patients to evaluate these CCR5 pooling patterns as they relate to progression and survival over 2 years.ResultsIn MB231 experiments, it was observed that CCR5 formed ~ 1 micron clusters identified as “CCR5 pools” on the surface of the cell, which in the presence of RANTES were endocytosed and translocated to the cell cytoplasm. When TACs from patients were analyzed, CCR5 pools were observed on the cell surface and translocating to the nuclear area, with CCR5 also having a positive statistical correlation between increased numbers of TACs and increased CCR5 pools on the cells. Further, it was determined that patients with very high numbers of CCR5 (> 10 CCR5 pools), specifically in the circulating stromal cells, were associated with worse progression-free survival (hazard ratio = 4.5, p = 0.002) and worse overall survival (hazard ratio = 3.7, p = 0.014).ConclusionsUsing a liquid biopsy approach, we evaluated two populations of tumor-associated cells emanating from primary tumors, with data suggesting that upregulation of the motility chemokine CCR5 in TACs provides clinically relevant opportunities for treating and tracking drug targetable receptors in mBC.

Multiple valence states of Fe boosting SERS activity of Fe3O4 nanoparticles and enabling effective SERS-MRI bimodal cancer imaging

Developing novel nanoparticle-based bioprobes utilized in clinical settings with imaging resolutions ranging from cell to tissue levels is a major challenge for tumor diagnosis and treatment. Herein, an optimized strategy for designing a Fe3O4-based bioprobe for dual-modal cancer imaging based on surface-enhanced Raman scattering (SERS) and magnetic resonance imaging (MRI) is introduced. Excellent SERS activity of ultrasmall Fe3O4 nanoparticles (NPs) was discovered, and a 5 × 10−9 M limit of detection for crystal violet molecules was successfully obtained. The high-efficiency interfacial photon-induced charge transfer in Fe3O4 NPs was promoted by multiple electronic energy levels ascribed to the multiple valence states of Fe, which was observed using ultraviolet–visible diffuse reflectance spectroscopy. Density functional theory calculations were utilized to reveal that the narrow band gap and high electron density of states of ultrasmall Fe3O4 NPs significantly boosted the vibronic coupling resonances in the SERS system upon illumination. The subtypes of cancer cells were accurately recognized via high-resolution SERS imaging in vitro using the prepared Fe3O4-based bioprobe with high sensitivity and good specificity. Notably, Fe3O4-based bioprobes simultaneously exhibited T1-weighted MRI contrast enhancement with an active targeting capability for tumors in vivo. To the best of our knowledge, this is the first report on the use of pure semiconductor-based SERS-MRI dual-modal nanoprobes in tumor imaging in vivo and in vitro, which has been previously realized only using semiconductor–metal complex materials. The non-metallic materials with SERS–MRI dual-modal imaging established in this report are a promising cancer diagnostic platform, which not only showed excellent performance in early tumor diagnosis but also possesses great potential for image-guided tumor treatment.

A racemosin B derivative, C25, suppresses breast cancer growth via lysosomal membrane permeabilization and inhibition of autophagic flux

Breast cancer is the most common malignancy among women worldwide. As conventional therapies are only partially successful in eradicating breast cancer, the development of novel strategies is a top priority. We previously showed that C25, a new racemosin B derivative, exerts its anti-cancer activity through inhibition of autophagy, but the underlying mechanism remained unknown. Here we show that C25 inhibits the growth of diverse breast cancer cell subtypes and effectively suppresses tumor progression in a xenotransplantation model of triple negative breast cancer. C25 acts as a lysosomotropic agent to induce lysosomal membrane permeabilization and inhibit autophagic flux, resulting in cathepsin release and cell death. In accordance, RNA sequencing and gene set enrichment analysis revealed that C25 induces pathways consistent with autophagy inhibition, cell cycle arrest and senescence. Interestingly, knockdown of TFEB or SQSTM1 reduced cell death induced by C25 treatment. Finally, we show that C25 synergizes with the chemo-therapeutics etoposide and paclitaxel to further limit breast cancer cell growth. Thus, C25 alone or in combination with other anti-neoplastic agents offers a novel therapeutic strategy for aggressive forms of breast cancer and possibly other malignancies.

Dinaciclib inhibits the stemness of two subtypes of human breast cancer cells by targeting the FoxM1 and Hedgehog signaling pathway.

Cyclin‑dependent kinase (CDK)4/6 inhibitors in combination with endocrine therapy are the current standard of care used in the first‑line treatment of hormone receptor‑positive/HER2‑negative metastatic breast cancer (BC). Although CDK4/6 inhibitors mainly target the cell cycle, emerging evidence has indicated further potential roles of CDKs other than regulating cell cycle progression. The G1 and G2/M transition regulators, including cyclins D and E, as well as their catalytic partners, CDK2, CDK4 and CDK6, have been reported to play crucial roles in pluripotency maintenance and cell fate decisions of human pluripotent stem cells by controlling transcription factors, signaling pathways and epigenetic regulators. Dinaciclib, a CDK1/2/5/9 inhibitor, is currently being evaluated in clinical trials against various cancer types, including BC. However, the underlying molecular mechanisms of CDK1/2/5/9 inhibitors in regulating BC stemness remain poorly understood. The present study aimed to examine the stemness‑inhibitory effects of dinaciclib in MCF‑7 (luminal) and HCC‑1806 (triple‑negative) BC cells. We found that this drug not only effectively reduced the self‑renewal abilities and other malignant properties, but also dose‑dependently decreased the protein expression levels of three BC stem cell markers, CD44, aldehyde dehydrogenase 1 family member A1 (ALDH1A1) and BMI1 proto‑oncogene, polycomb ring finger (Bmi1), as well as three embryonic stem cell markers, Oct4, Nanog and Sox2. Moreover, the dinaciclib‑induced decrease of Oct4 and Nanog protein expression was able to be restored by co‑treatment with MG‑132, a proteasome inhibitor. Forkhead box M1 (FoxM1), both a stemness‑stimulating transcription factor and a cell cycle regulator, along with the Hedgehog signaling pathway, were identified as the therapeutic targets of dinaciclib. Collectively, the present results demonstrated a novel role of dinaciclib in suppressing BC stemness and indicated its potential use for future cancer treatments.

Assessment of Pharmacological Interactions between SIRT2 Inhibitor AGK2 and Paclitaxel in Different Molecular Subtypes of Breast Cancer Cells.

Breast carcinoma (BC) is the most commonly diagnosed type of cancer in women in the world. Although the advances in the treatment of BC patients are significant, numerous side effects, severe toxicity towards normal cells as well as the multidrug resistance (MDR) phenomenon restrict the effectiveness of the therapies used. Therefore, new active compounds which decrease the MDR, extend disease-free survival, thereby ameliorating the effectiveness of the current treatment regimens, are greatly needed. Histone deacetylase inhibitors (HDIs), including sirtuin inhibitors (SIRTi), are the epigenetic antitumor agents which induce a cytotoxic effect in different types of cancer cells, including BC cells. Currently, combined forms of therapy with two or even more chemotherapeutics are promising antineoplastic tools to obtain a better response to therapy and limit adverse effects. Thus, on the one hand, much more effective chemotherapeutics, e.g., sirtuin inhibitors (SIRTi), are in demand; on the other hand, combinations of accepted cytostatics are trialed. Thus, the aim of our research was to examine the combination effects of a renowned cytotoxic drug paclitaxel (PAX) and SIRT2 inhibitor AGK2 on the proliferation and viability of the T47D, MCF7, MDA-MB-231, MDA-MB-468, BT-549 and HCC1937 BC cells. Moreover, cell cycle arrest and apoptosis induction were explored. The type of pharmacological interactions between AGK2 and PAX in different molecular subtypes of BC cells was assessed using the advanced isobolographic method. Our findings demonstrated that the tested active agents singly inhibited viability and proliferation of BC cells as well as induced cell cycle arrest and apoptosis in the cell-dependent context. Additionally, AGK2 increased the antitumor effect of PAX in most BC cell lines. We observed that, depending on the BC cell lines, the combinations of tested drugs showed synergistic, additive or antagonistic pharmacological interaction. In conclusion, our studies demonstrated that the consolidated therapy with the use of AGK2 and PAX can be considered as a potential therapeutic regimen in the personalized cure of BC patients in the future.

In situ ratiometric SERS imaging of intracellular protease activity for subtype discrimination of human breast cancer

Accurate discrimination between different cells at the molecular level is of fundamental importance for disease diagnosis. Endogenous proteases are such molecular candidates for cancer cell subtype study. But in situ probing their activity in live cells remains challenging for surface-enhanced Raman scattering (SERS). Here, we present a sensitive ratio-type SERS nanoprobe for imaging of matrix metalloproteinase-2 (MMP-2) in different cancer cells subtypes. The nanoprobe contained three components: a plasmon-active gold nanoparticle as the SERS enhancing matrix, Raman dye rhodamine B (Rh B)-labelled substrate peptides as the specific MMP-2 recognizer, and 2-naphthalenethiol (2-NT) as the internal standard. MMP-2-responsive cleavage of peptides from the nanoprobe surface results in decrease or even disappearance of SERS emission of Rh B, which was ratioed over the emission of 2-NT for the quantification of MMP-2 activity. Both in-tube assay and in-cell imaging results show that the MMP-responsive nanoprobe can work and serve to differentiate the normal breast cells from the tumorous ones, to differentiate two breast cancer cell subtypes with a different degree of malignancy. We believe that this SERS nanoprobe could find a wide application in the fields of tumor biology and accurate disease diagnosis.

Accurate Tumor Subtype Detection with Raman Spectroscopy via Variational Autoencoder and Machine Learning.

Accurate diagnosis of cancer subtypes is a great guide for the development of surgical plans and prognosis in the clinic. Raman spectroscopy, combined with the machine learning algorithm, has been demonstrated to be a powerful tool for tumor identification. However, the analysis and classification of Raman spectra for biological samples with complex compositions are still challenges. In addition, the signal-to-noise ratio of the spectra also influences the accuracy of the classification. Herein, we applied the variational autoencoder (VAE) to Raman spectra for downscaling and noise reduction simultaneously. We validated the performance of the VAE algorithm at the cellular and tissue levels. VAE successfully downscaled high-dimensional Raman spectral data to two-dimensional (2D) data for three subtypes of non-small cell lung cancer cells and two subtypes of kidney cancer tissues. Gaussian naïve bayes was applied to subtype discrimination with the 2D data after VAE encoding at both the cellular and tissue levels, significantly outperforming the discrimination results using original spectra. Therefore, the analysis of Raman spectroscopy based on VAE and machine learning has great potential for rapid diagnosis of tumor subtypes.

Synthesis, anticancer and antimalarial activities of organosulfur and organoselenium derivatives of cyclopentadienyliron dicarbonyl as photoCORMs

The reactions of [CpFe(CO)2]2 with 2 mol equivalents of R2E2 (R = C6H11, C4H3S, CH2C6H5; E = S, Se) have yielded dinuclear products of [CpFe(CO)(μ-L)]2 (L = C6H11S (1), C4H3S2 (2) and CH2C6H5Se (3)), respectively. An additional trinuclear compound, C24H22O2Se2Fe3 (4) was isolated only from the reaction with dibenzyl diselenide. All the products have been fully characterized by FT-IR,1H and13C NMR, CHN elemental analysis, and structurally elucidated by single crystal X-ray diffraction. The stability of 1–3 both under dark and UV conditions were investigated by monitoring the changes in their respective UV–Vis spectra in phosphate buffered saline (PBS) solution. Their potential application as photoCORMs were also evaluated by standard myoglobin assay. 2 showed the shortest half-life and highest equivalent of CO-released. The anticancer studies of 1–3 were evaluated in-vitro on breast cancer cell lines (MCF-7, MDA-MB-231 and MDA-MB-468) and the cytotoxicity of 2 had increased approximately 4 times against MDA-MB-468 cell line upon UV irradiation, which was 11.5 ± 2.7 μM. All complexes exhibited cytotoxic effect on specific sub-types of breast cancer cells. Antimalarial activities of 1–3 had also been studied and reported in this work. Unfortunately, these complexes did not display any significant effect on the Plasmodium falciparum 3D7 strains.

ANALYSIS OF RADON GAS EXPOSURE IN THE HOME ON THE RISK OF LUNG CANCER (NARRATIVE REVIEW)

According to WHO 2009, exposure to radon gas is known to be the second highest cause of lung cancer. The United States Environmental Protection Agency (U.S.EPA) estimates that 21,000 lung cancer deaths annually result from exposure to radon in the United States. Radon (ð‘…ð‘›222) naturally originates in soil and rock and it is the decay of Uranium-238 (𑈠238). This study used a literature review study that aims to review several research journal articles regarding exposure to Radon gas in the home with the risk of lung cancer. The data base used to search for research journal articles are Science Direct, PubMed, DOAJ, JSTOR, and Taylor & Francis. The results of this study were: (1) nine out of twelve journal articles described a significant association between Rn in the home (concentration of Rn 37 Bq/ð‘š3 until ≥ 300 Bq/ð‘š3 ) and the risk of lung cancer; (2) the duration of stay of the occupants in the house lasted from 2-30 years; and (3) eight journal articles presented that the most common lung cancer cell subtype associated with Rn exposure in the home was adenocarcinoma and squamous cell carcinoma. People need to maintain indoor airflow and choosing safe building material (not containing Rn).

A novel long non-coding RNA regulates the integrin, ITGA2 in breast cancer.

ITGA2 encodes the integrin, α2 which mediates metastatic progression, and is a predictor of poor prognosis and chemoresistance in breast cancer. Decreased ITGA2 promoter methylation is implicated as a driver of increased gene expression in aggressive prostate and pancreatic tumours, however the contribution of altered methylation to ITGA2 expression changes in breast tumours has not been examined. ITGA2 gene methylation and gene expression was examined in publicly available breast cancer datasets, and ITGA2 promoter methylation was mapped by targeted bisulphite sequencing analysis in breast tumour cell lines. The expression of a putative regulatory long noncoding RNA (lncRNA) was examined by qPCR and its' functionality was investigated using gene knockdown (antisense oligonucleotides) and over expression in breast cancer cell lines. In breast tumours and breast cancer cell lines the ITGA2 promoter is largely unmethylated, with gene expression variable in tumour subtypes, irrespective of promoter methylation. A novel lncRNA (AC025180.1;ENSG00000249899), named herein I2ALR, was identified at the ITGA2 gene locus, and was variably expressed in breast tumours and breast cancer cell subtypes. I2LAR knockdown resulted in upregulation of ITGA2 gene expression, whilst over-expression of I2ALR resulted in downregulation of ITGA2 mRNA. Further, examination of two downstream targets of ITGA2 associated with breast tumor stemness and metastasis (CCND1 and ACLY), revealed concomitant gene expression changes in response to I2ALR modulation. I2ALR represents a novel regulatory molecule targeting ITGA2 expression in breast tumours; a finding of significant and topical interest to the development of therapeutics targeting this integrin.

Interaction between prostate cancer stem cells and bone microenvironment regulates prostate cancer bone metastasis and treatment resistance.

Prostate cancer (PCa) is one of the most common cancers with increasing rates of incidence. Bone metastasis and drug resistance are the most serious threat faced by patients following a delayed diagnosis of cancer, which might lead to treatment failure and death. The theoretical model of cancer stem cells (CSCs) explains the diverse molecular characteristics of cancer as well as its relapse, metastasis and drug resistance. Prostate cancer involves heterogeneous cells community, including prostate cancer stem cells as an important component. These subtypes of cancer cells are usually monoclonal, expressing specific biomarkers and exhibiting self-renewal and differentiation capacity. Therefore, therapies that target CSCs might be more effective in overcome drug resistance and metastasis. Thus, anti-CSCs therapies differ from the traditional anti-proliferative approach. We focus here on reviewing the effects of prostate CSCs on bone metastasis and resistance to traditional treatment in PCa, and report new clinical strategies that address CSC-based tumorigenesis.

RNF12 is regulated by AKT phosphorylation and promotes TGF-\u03b2 driven breast cancer metastasis

Transforming growth factor-β (TGF-β) acts as a pro-metastatic factor in advanced breast cancer. RNF12, an E3 ubiquitin ligase, stimulates TGF-β signaling by binding to the inhibitory SMAD7 and inducing its proteasomal degradation. How RNF12 activity is regulated and its exact role in cancer is incompletely understood. Here we report that RNF12 was overexpressed in invasive breast cancers and its high expression correlated with poor prognosis. RNF12 promoted breast cancer cell migration, invasion, and experimental metastasis in zebrafish and murine xenograft models. RNF12 levels were positively associated with the phosphorylated AKT/protein kinase B (PKB) levels, and both displayed significant higher levels in the basal-like subtype compared with the levels in luminal-like subtype of breast cancer cells. Mechanistically, AKT-mediated phosphorylation induced the nuclear localization of RNF12, maintained its stability, and accelerated the degradation of SMAD7 mediated by RNF12. Furthermore, we demonstrated that RNF12 and AKT cooperated functionally in breast cancer cell migration. Notably, RNF12 expression strongly correlated with both phosphorylated AKT and phosphorylated SMAD2 levels in breast cancer tissues. Thus, our results uncovered RNF12 as an important determinant in the crosstalk between the TGF-β and AKT signaling pathways during breast cancer progression.

Single cell multi-miRNAs quantification with hydrogel microbeads for liver cancer cell subtypes discrimination.

The simultaneous quantification of multi-miRNAs in single cells reveals cellular heterogeneity, and benefits the subtypes discrimination of cancer cells . Though micro-droplet techniques enable successful single cell encapsulation, the isolated and restricted reaction space of microdroplets causes cross-reactions and inaccuracy for simultaneous multi-miRNAs quantification. Herein, we develop a hydrogel microbead based strategy for the simultaneous sensitive quantification of miRNA-21, 122 and 222 in single cells. Single cells are encapsulated and undergo cytolysis in hydrogel microbeads. The three target miRNAs are retained in the microbead by pre-immobilized capture probes, and activate rolling circle amplification (RCA) reactions. The RCA products are hybridized with corresponding dye labelled DNA reporters, and the respective fluorescence intensities are recorded for multi-miRNA quantification. The porous structure of the hydrogel microbeads allows the free diffusion of reactants and easy removal of unreacted DNA strands, which effectively avoids nonspecific cross-reactions. Clear differentiation of cellular heterogeneity and subpopulation discrimination are achieved for three kinds of liver cancer cells and one normal liver cell.

Breast Cancer Cell Subtypes Display Different Metabolic Phenotypes That Correlate with Their Clinical Classification.

Simple SummaryRecent studies on cancer cell metabolism have achieved notable breakthroughs that have led to a new scientific paradigm. How cancer cell metabolic reprogramming is orchestrated and the decisive role of this reprogramming in the oncogenic process and tumor adaptative evolution has been characterized at the molecular level. Despite this knowledge, it is essential to understand how cancer cells can metabolically respond as a living whole to ensure their survival and adaptation potential. In this work, we investigated whether different cancers and different subtypes display different metabolic phenotypes with a focus on breast cancer cell models representative of each clinical subtype. The potential results might have significant translational implications for diagnostic, prognostic and therapeutic applications.Metabolic reprogramming of cancer cells represents an orchestrated network of evolving molecular and functional adaptations during oncogenic progression. In particular, how metabolic reprogramming is orchestrated in breast cancer and its decisive role in the oncogenic process and tumor evolving adaptations are well consolidated at the molecular level. Nevertheless, potential correlations between functional metabolic features and breast cancer clinical classification still represent issues that have not been fully studied to date. Accordingly, we aimed to investigate whether breast cancer cell models representative of each clinical subtype might display different metabolic phenotypes that correlate with current clinical classifications. In the present work, functional metabolic profiling was performed for breast cancer cell models representative of each clinical subtype based on the combination of enzyme inhibitors for key metabolic pathways, and isotope-labeled tracing dynamic analysis. The results indicated the main metabolic phenotypes, so-called ‘metabophenotypes’, in terms of their dependency on glycolytic metabolism or their reliance on mitochondrial oxidative metabolism. The results showed that breast cancer cell subtypes display different metabophenotypes. Importantly, these metabophenotypes are clearly correlated with the current clinical classifications.

Circular RNA circPIP5K1A contributes to cancer stemness of osteosarcoma by miR-515-5p/YAP axis

BackgroundOsteosarcoma is a common type of bone tumors and frequently occurs in children and adolescents. Cancer stem cells (CSCs) are a unique sub-type of self-renewal cancer cells and the stemness of cancer cells are involved in the spread, recurrence, metastasis, and even therapeutic resistance. However, the regulation mechanisms of CSCs in osteosarcoma are poorly understood. Circular RNA (circRNA) is a unique sort of non-coding RNAs and widely participate in the modulation of cancer progression.MethodsIn this study, we identified the critical function of circular RNA circPIP5K1A in stemness of osteosarcoma cells.ResultsCircPIP5K1A expression was significantly enhanced in clinical osteosarcoma tissues compared with the adjacent normal tissues. The depletion of circPIP5K1A by siRNA repressed osteosarcoma cell viabilities and induced osteosarcoma cell apoptosis. The suppression of circPIP5K1A attenuated the capabilities of invasion and migration of osteosarcoma cells. The circPIP5K1A knockdown increased E-Cadherin expression and decreased Vimentin expression in osteosarcoma cells. The sphere formation abilities of osteosarcoma cells were repressed by the depletion of circPIP5K1A. The CD133+CD44+ cell population of osteosarcoma cells was reduced by circPIP5K1A knockdown. The expression of ALDH1 and Nanog was decreased by the inhibition of circPIP5K1A in osteosarcoma cells. Mechanically, circPIP5K1A enhanced YAP expression by targeting miR-515-5p. MiR-515-5p inhibited stemness of osteosarcoma cells. The CSCs properties of osteosarcoma cells were repressed by circPIP5K1A knockdown or miR-515-5p mimic, while miR-515-5p inhibitor or YAP overexpression reversed circPIP5K1A knockdown-induced repression. Tumor xenograft analysis in nude mice demonstrated that the depletion of circPIP5K1A represses osteosarcoma cell growth in vivo.ConclusionIn conclusion, we identified that circular RNA circPIP5K1A contributed to cancer stemness of osteosarcoma by miR-515-5p/YAP axis. Targeting circPIP5K1A may be considered as a potential therapeutic strategy for osteosarcoma treatment.

Network Pharmacology-Based Identification of Key Mechanisms of Xihuang Pill in the Treatment of Triple-Negative Breast Cancer Stem Cells.

Xihuang pill, an approved Chinese medicine formula (state medical permit number. Z11020073), is a commonly used adjuvant drug for cancer patients in China. Xihuang pill has a satisfactory effect in treating breast cancer in clinics, especially triple-negative breast cancer (TNBC), which is the most aggressive type of breast cancer, and finite effective therapies. However, the mechanism of Xihuang pill in treating TNBC remains unclear. The present study aims to explore the pharmacological mechanism of Xihuang pill in treating advanced TNBC. We identified the main chemical components of Xihuang pill by using HPLC-Q-TOF-MS/MS. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis shows that serum containing Xihuang pill (XS) had no obvious killing effect on any subtype of breast cancer cells, but it inhibited mammosphere colony formation of two TNBC cell lines (4T1 and HCC1806 cells) and could enhance the inhibitory effect of paclitaxel (PTX) on the proliferation of 4T1 and HCC1806 cells when combined with PTX. Seventy-six active compounds in Xihuang pill, their 300 protein targets, and 16667 TNBC stem cell–related genes were identified. The drug–herb–active compound–target gene–disease network and enrichment analyses were constructed with 190 overlapping candidate targets. Through text mining and molecular docking, the target gene NR3C2 and its active compound naringenin were selected for further validation. According to the TCGA database, we observed that a high expression of NR3C2 promoted a higher survival probability regarding overall survival (OS). In vitro experiments indicated that naringenin presented an identical effect to XS, possibly by regulating the NR3C2 expression. Overall, this study explored the effect of Xihuang pill in treating advanced TNBC cells and showed that naringenin, which is the key active compound of Xihuang pill, could lessen the stemness of TNBC cells to produce a synergistic effect on PTX by regulating the NR3C2 gene.

Characterizing Intercellular Communication of Pan-Cancer Reveals SPP1+ Tumor-Associated Macrophage Expanded in Hypoxia and Promoting Cancer Malignancy Through Single-Cell RNA-Seq Data.

Hypoxia is a characteristic of tumor microenvironment (TME) and is a major contributor to tumor progression. Yet, subtype identification of tumor-associated non-malignant cells at single-cell resolution and how they influence cancer progression under hypoxia TME remain largely unexplored. Here, we used RNA-seq data of 424,194 single cells from 108 patients to identify the subtypes of cancer cells, stromal cells, and immune cells; to evaluate their hypoxia score; and also to uncover potential interaction signals between these cells in vivo across six cancer types. We identified SPP1+ tumor-associated macrophage (TAM) subpopulation potentially enhanced epithelial–mesenchymal transition (EMT) by interaction with cancer cells through paracrine pattern. We prioritized SPP1 as a TAM-secreted factor to act on cancer cells and found a significant enhanced migration phenotype and invasion ability in A549 lung cancer cells induced by recombinant protein SPP1. Besides, prognostic analysis indicated that a higher expression of SPP1 was found to be related to worse clinical outcome in six cancer types. SPP1 expression was higher in hypoxia-high macrophages based on single-cell data, which was further validated by an in vitro experiment that SPP1 was upregulated in macrophages under hypoxia-cultured compared with normoxic conditions. Additionally, a differential analysis demonstrated that hypoxia potentially influences extracellular matrix remodeling, glycolysis, and interleukin-10 signal activation in various cancer types. Our work illuminates the clearer underlying mechanism in the intricate interaction between different cell subtypes within hypoxia TME and proposes the guidelines for the development of therapeutic targets specifically for patients with high proportion of SPP1+ TAMs in hypoxic lesions.

DNA Logic Circuits for Multiple Tumor Cells Identification Using Intracellular MicroRNA Molecular Bispecific Recognition.

The aberrant expression level of intracellular microRNAs (miRNAs) holds great promise for differentiating cell types at the molecular level. However, cell subtype discrimination based on a single miRNA molecular level is not sufficient and reliable. Herein, multiple identifiable and reporting modules are integrated into a single DNA circuit for multiple cancer cell subtypes identification based on miRNAs bispecific recognition. The DNA three-dimensional (3D) logic gate nano-hexahedron framework extends three recognition modules and three reporting modules to form three "AND" logic gates. Each Boolean operator "AND" returns an "ON" signal in the presence of bispecific miRNAs, simultaneously enabling three types of cell subtype identification. It not only enables the discrimination of cancer cells A549 and MCF-7 from normal cells NHDF but also successfully distinguishes different types of cancer cells. The bispecific intracellular miRNA controllable DNA circuit, with low signal-to-noise ratio, easily extends to various cell type discrimination by adjusting the miRNA species, provides huge opportunities for accurately differentiating multiple cell types at the molecular level.

Discovery and construction of prognostic model for clear cell renal cell carcinoma based on single-cell and bulk transcriptome analysis.

BackgroundClear cell renal cell carcinoma (ccRCC) is the most common malignant kidney tumor in adults. Single-cell transcriptome sequencing can provide accurate gene expression data of individual cells. Integrated single-cell and bulk transcriptome data from ccRCC samples provide comprehensive information, which allows the discovery of new understandings of ccRCC and the construction of a novel prognostic model for ccRCC patients.MethodsSingle-cell transcriptome sequencing data was preprocessed by using the Seurat package in R software. Principal component analysis (PCA) and the t-distributed stochastic neighbor embedding (t-SNE) algorithm were used to perform cluster classification. Two subtypes of cancer cells were identified, pseudotime trajectory analysis and gene ontology (GO) analysis were conducted with the monocle and clusterProfiler packages. Two novel cancer cell biomarkers were identified according to the single-cell sequencing and were confirmed by The Cancer Genome Atlas (TCGA) data. T cell-related marker genes according to single-cell sequencing were screened by a combination of Kaplan-Meier (KM) analysis, univariate Cox analysis, least absolute shrinkage and selection operator (Lasso) regression and multivariate Cox analysis of TCGA data. Four survival predicting genes were screened out to develop a risk score model. A nomogram consisting of the risk score and clinical information was constructed to predict the prognosis for ccRCC patients.ResultsA total of 5,933 cells were included in the study after quality control. Fifteen cell clusters were classified by PCA and t-SNE algorithm. Two clusters of cancer cells with distinct differentiation status were identified. Besides, GO analysis revealed that biological processes were different between the two subgroups. Egl-9 family hypoxia-inducible factor 3 (EGLN3) and nucleolar protein 3 (NOL3) were specifically expressed in cancer cell clusters, bulk RNA sequencing data from TCGA confirmed their high expression in ccRCC tissues. GTSE1, CENPF, SMC2 and H2AFV were screened out and applied to the construction of risk score model. A nomogram was generated to predict prognosis of ccRCC by combing the risk score and clinical parameters.ConclusionsWe integrated single-cell and bulk transcriptome data from ccRCC in this study. Two subtypes of ccRCC cells with different biological characteristics and two potential biomarkers of ccRCC were discovered. A novel prognostic model was constructed for clinical application.