Enrichment Of Cancer Stem Cells Research Articles

Effect of microserum environment stimulation on extraction and biological function of colorectal cancer stem cells

Background3D cancer stem cell (CSC) cultures are widely used as in vitro tumor models. In this study, we determined the effects of enriching HCT116 tumor spheres initially cultured in serum-free medium with different concentrations of serum, focusing on the effect of microserum environment stimulation on extraction and biological function of colorectal cancer stem cells (CCSCs).MethodsCCSCs were enriched in standard serum-free medium and serum-free medium with different concentrations of serum for 1 week. The expression of CSC-associated markers in CCSCs, and the presence and relative proportion of CSCs (CD133/CD44 cell sorting) were then determined to elucidate the effect of the microserum environment on the preservation of CSC-related features. Further, the tumorigenic capacity of CCSCs was evaluated in an immunodeficiency mouse model.ResultsOur data indicated that a significantly greater number of spheres with a greater size range and high viability without drastic alteration in biological and structural features, which maintained self‐renewal potential after sequential passages were formed after serum supplementation. Real-time analysis showed that both serum spheres and serum-free spheres displayed similar expression patterns for key stemness genes. Serum spheres showed higher expression of the CSC surface markers CD133 and CD44 than did CSCs spheres cultured in serum-free medium. Adherent cultures in complete medium could adapt to the serum-containing microenvironment faster and showed higher proliferation ability. The addition of serum induced EMT and promoted the migration and invasion of serum globular cells. Compared with serum-free cells and adherent cells, serum spheres showed higher tumor initiation ability.ConclusionsMicroserum environment stimulation could be an effective strategy for reliable enrichment of intact CCSCs, and a more efficient CSC enrichment method.

Influence of Bruton's Tyrosine Kinase (BTK) on Epithelial-Mesenchymal Transition (EMT) Processes and Cancer Stem Cell (CSC) Enrichment in Head and Neck Squamous Cell Carcinoma (HNSCC).

Constitutively active kinases play a crucial role in carcinogenesis, and their inhibition is a common target for molecular tumor therapy. We recently discovered the expression of two oncogenic isoforms of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC), Btk-p80 and BTK-p65. However, the precise role of BTK in HNSCC remains unclear. Analyses of a tissue microarray containing benign and malignant as well as inflammatory tissue samples of the head and neck region revealed the preferential expression of BTK-p80 in malignant tissue, whereas BTK-p65 expression was confirmed in over 80% of analyzed metastatic head and neck tumor cases. Therefore, processes associated with metastasis, like cancer stem cell (CSC) enrichment and the epithelial-mesenchymal transition (EMT), which in turn depend on an appropriate cytokine milieu, were analyzed. Treatment of HNSCC-derived cell lines cultured under 3D conditions with the BTK inhibitor AVL-292 caused reduced sphere formation, which was accompanied by reduced numbers of ALDH1A1+ CSCs as well as biological changes associated with the EMT. Moreover, we observed reduced NF-κB expression as well as altered NF-κB dependent pro-tumorigenic and EMT-associated cytokine release of IL-6, IFNγ, and TNFα when BTK activity was dampened. Therefore, an autocrine regulation of the oncogenic BTK-dependent process in HNSCC can be suggested, with BTK inhibition expected to be an effective treatment option for HNSCC.

Peristalsis-Associated Mechanotransduction Drives Malignant Progression of Colorectal Cancer.

In the colorectal cancer (CRC) tumor microenvironment, cancerous and precancerous cells continuously experience mechanical forces associated with peristalsis. Given that mechanical forces like shear stress and strain can positively impact cancer progression, we explored the hypothesis that peristalsis may also contribute to malignant progression in CRC. We defined malignant progression as enrichment of cancer stem cells and the acquisition of invasive behaviors, both vital to CRC progression. We leveraged our peristalsis bioreactor to expose CRC cell lines (HCT116), patient-derived xenograft (PDX1,2) lines, or non-cancerous intestinal cells (HIEC-6) to forces associated with peristalsis in vitro. Cells were maintained in static control conditions or exposed to peristalsis for 24 h prior to assessment of cancer stem cell (CSC) emergence or the acquisition of invasive phenotypes. Exposure of HCT116 cells to peristalsis significantly increased the emergence of LGR5+ CSCs by 1.8-fold compared to static controls. Peristalsis enriched LGR5 positivity in several CRC cell lines, notably significant in KRAS mutant lines. In contrast, peristalsis failed to increase LGR5+ in non-cancerous intestinal cells, HIEC-6. LGR5+ emergence downstream of peristalsis was dependent on ROCK and Wnt activity, and not YAP1 activation. Additionally, HCT116 cells adopted invasive morphologies when exposed to peristalsis, with increased filopodia density and epithelial to mesenchymal gene expression, in a Wnt dependent manner. Peristalsis associated forces drive malignant progression of CRC via ROCK, YAP1, and Wnt-related mechanotransduction. The online version contains supplementary material available at 10.1007/s12195-023-00776-w.

Hypoxia: syndicating triple negative breast cancer against various therapeutic regimens.

Triple-negative breast cancer (TNBC) is one of the deadliest subtypes of breast cancer (BC) for its high aggressiveness, heterogeneity, and hypoxic nature. Based on biological and clinical observations the TNBC related mortality is very high worldwide. Emerging studies have clearly demonstrated that hypoxia regulates the critical metabolic, developmental, and survival pathways in TNBC, which include glycolysis and angiogenesis. Alterations to these pathways accelerate the cancer stem cells (CSCs) enrichment and immune escape, which further lead to tumor invasion, migration, and metastasis. Beside this, hypoxia also manipulates the epigenetic plasticity and DNA damage response (DDR) to syndicate TNBC survival and its progression. Hypoxia fundamentally creates the low oxygen condition responsible for the alteration in Hypoxia-Inducible Factor-1alpha (HIF-1α) signaling within the tumor microenvironment, allowing tumors to survive and making them resistant to various therapies. Therefore, there is an urgent need for society to establish target-based therapies that overcome the resistance and limitations of the current treatment plan for TNBC. In this review article, we have thoroughly discussed the plausible significance of HIF-1α as a target in various therapeutic regimens such as chemotherapy, radiotherapy, immunotherapy, anti-angiogenic therapy, adjuvant therapy photodynamic therapy, adoptive cell therapy, combination therapies, antibody drug conjugates and cancer vaccines. Further, we also reviewed here the intrinsic mechanism and existing issues in targeting HIF-1α while improvising the current therapeutic strategies. This review highlights and discusses the future perspectives and the major alternatives to overcome TNBC resistance by targeting hypoxia-induced signaling.

Loss of function of GATA3 regulates FRA1 and c-FOS to activate EMT and promote mammary tumorigenesis and metastasis

Basal-like breast cancers (BLBCs) are among the most aggressive cancers, partly due to their enrichment of cancer stem cells (CSCs). Breast CSCs can be generated from luminal-type cancer cells via epithelial-mesenchymal transition (EMT). GATA3 maintains luminal cell fate, and its expression is lost or reduced in BLBCs. However, deletion of Gata3 in mice or cells results in early lethality or proliferative defects. It is unknown how loss-of-function of GATA3 regulates EMT and CSCs in breast cancer. We report here that haploid loss of Gata3 in mice lacking p18Ink4c, a cell cycle inhibitor, up-regulates Fra1, an AP-1 family protein that promotes mesenchymal traits, and downregulates c-Fos, another AP-1 family protein that maintains epithelial fate, leading to activation of EMT and promotion of mammary tumor initiation and metastasis. Depletion of Gata3 in luminal tumor cells similarly regulates Fra1 and c-Fos in activation of EMT. GATA3 binds to FOSL1 (encoding FRA1) and FOS (encoding c-FOS) loci to repress FOSL1 and activate FOS transcription. Deletion of Fra1 or reconstitution of Gata3, but not reconstitution of c-Fos, in Gata3 deficient tumor cells inhibits EMT, preventing tumorigenesis and/or metastasis. In human breast cancers, GATA3 expression is negatively correlated with FRA1 and positively correlated with c-FOS. Low GATA3 and FOS, but high FOSL1, are characteristics of BLBCs. Together, these data provide the first genetic evidence indicating that loss of function of GATA3 in mammary tumor cells activates FOSL1 to promote mesenchymal traits and CSC function, while concurrently repressing FOS to lose epithelial features. We demonstrate that FRA1 is required for the activation of EMT in GATA3 deficient tumorigenesis and metastasis.

Chemotherapy induces breast cancer stem cell enrichment through repression of glutathione S-transferase Mu

Chemotherapy induces breast cancer stem cell enrichment through repression of glutathione S-transferase Mu

Amino acid profile changes during enrichment of spheroid cells with cancer stem cell properties in MCF-7 and MDA-MB-231 cell lines.

Cancer stem cells (CSCs), subpopulations of cancer cells, are responsible for tumor progression, metastasis, and relapse. Changes in amino acid metabolism are linked to breast cancer recurrence and metastasis. This study aimed to evaluate the changes in the amino acid profile in MCF-7 and MDA-MB-231 cells during spheroid formation to discover the specific metabolic properties in CSCs. MCF-7 and MDA-MB-231 breast cancer cells were cultured as spheroids and evaluated to characterize their CSC properties. The characteristics of CSC were evaluated by examining the expression of CSC markers and conducting drug resistance assays. In addition, amino acid profile change during the enrichment of breast cancer stem cells in the spheroids was investigated by high-performance liquid chromatography (HPLC). The results indicated that out of 20 different amino acids analyzed, 19 of them decreased during the spheroid formation process. Alanine, lysine, phenylalanine, threonine, and glycine showed significant reductions in the conditioned media of both cell lines in the spheroid form compared to the monolayer cells. Only one of the amino acids increased in MCF-7 and MDA-MB-231 spheroids (histidine and serine, respectively). Our results suggest that certain amino acids identified in this study can be used for a better understanding of the molecular mechanisms associated with breast cancer stem cell formation.

A Tumor Microenvironment-Driven Network Regulated by STAT3 and p65 Negatively Controls the Enrichment of Cancer Stem Cells in Human HR+/HER2− Breast Cancer

Hormone receptor-positive and HER2-negative (HR+/HER2-; luminal A) tumors are prevalent in breast cancer. Our past studies demonstrated that "TME Stimulation" (estrogen + TNFα + EGF, representing three arms of the tumor microenvironment, TME) has enriched metastasis-forming cancer stem cells (CSCs) in HR+/HER2- human breast cancer cells. Here, following information obtained by RNAseq analyses of TME-stimulated CSCs and Non-CSCs, we found that TME Stimulation has induced the activation of S727-STAT3, Y705-STAT3, STAT1 and p65. Upon TME Stimulation, stattic (STAT3 inhibitor) usage demonstrated that Y705-STAT3 activation negatively controlled CSC enrichment and epithelial-to-mesenchymal transition (EMT) traits, while inducing CXCL8 (IL-8) and PD-L1 expression. However, STAT3 knock-down (siSTAT3) had no effect on these functions; in terms of CSC enrichment, p65 had down-regulatory roles that compensated for the loss of an entire STAT3 protein. Y705-STAT3 and p65 acted additively in reducing CSC enrichment, and Y705A-STAT3 variant + sip65 has enriched chemo-resistant CSCs. Clinical data analyses revealed an inverse correlation between Y705-STAT3 + p65 phosphorylation and CSC signature in luminal A patients, and connection to improved disease course. Overall, we find regulatory roles for Y705-STAT3 and p65 in TME-stimulated HR+/HER2- tumors, with the ability to limit CSC enrichment. These findings raise concerns about using inhibitors of STAT3 and p65 as therapeutic strategies in the clinic.

Abstract 5804: Targeting methyl donor synthesis inhibits platinum-induced enrichment of ovarian cancer stem cells

Abstract Ovarian cancer (OC) is one of the deadliest gynecologic cancers in US. Platinum (Pt)-based chemotherapy can reprogram cancer cells to ovarian cancer stem cells (OCSCs) and contribute to incurable recurrence. We have previously shown that Pt-containing drugs, e.g., cisplatin, enrich for OCSCs and Pt-induced alterations in DNA methylation enhanced OCSC malignant properties. CSCs have been shown to be dependent on methionine (Met) in many cancers, and in the current study, we investigated the role of Met and the interplay between methylation and Met metabolism in OCSC enrichment. In high grade serous ovarian cancer cell lines (OVCAR5, OVCAR3), cisplatin treatment (IC50, 16hr) increased (p<0.01) the percentage of cells that were positive for aldehyde dehydrogenase (ALDH+ cells), a known OCSCs marker. Cisplatin treatment also increased (p<0.05) the whole cell level of 5-methylcytosine, indicating Pt-induced methylation changes. In addition, Met metabolism was altered by cisplatin, demonstrated by decreased (p<0.01) level of Met derivative, S-adenosyl-methionine (SAM), and increased (p<0.01) expression of Met utilization enzyme, methionine adenosyltransferase 2A (MAT2A). To investigate a possible role of Met metabolism in Pt-induced enrichment of OCSCs, OVCAR5 cells were cultured in complete or Met-depleted (Met-) media, treated with cisplatin or vehicle and examined for CSC properties, including ALDH+ cells and the ability to form spheroids under low-attachment conditions. Met depletion blocked (p<0.01) both the Pt-induced increase of ALDH+ cells and spheroid formation. Both Met- only and Met- in combination with cisplatin activated p38, determined by increased phosphorylated p38, which may have responded to Met depletion and promoted cell cycle arrest. In addition, MAT2A expression was higher (p<0.01) in ALDH+ compared to ALDH- OC cells. MAT2A inhibitor FIDAS-5 combined with cisplatin blocked the Pt-induced increase in ALDH+ cells (p<0.01), demonstrating a requirement for Met utilization in Pt-induced OCSC enrichment. As MAT2A is a therapeutic target in cancer, MAT2A inhibition could represent a therapeutic approach for inhibiting CSC in ovarian cancer. We are further investigating the mechanism of MAT2A-mediated inhibition on Pt-induced OCSCs enrichment by examining the OC methylome and cell cycle arrest. Citation Format: Shu Zhang, Tara X. Metcalfe, Christiane A. Hassel, Heather M. O'Hagan, Kenneth P. Nephew. Targeting methyl donor synthesis inhibits platinum-induced enrichment of ovarian cancer stem cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5804.

Abstract 5809: Tracking cancer stem cell expression of pancreatic and ampullary cancer organoids in response to chemotherapy

Abstract Background: Cancer stem cells (CSCs) have been described to confer resistance and aggressive disease biology in pancreatic ductal adenocarcinoma (PDAC) and ampullary carcinoma (AMPC). Patient derived cancer organoids (PCOs) are an important model of PDAC that maintain the molecular features of advanced disease and have been shown to predict drug response. This study aimed to determine the dynamics of CSC expression on the oligoclonal culture growth of three independent PCOs models. Methods: PCOs were assayed using low volume 10μL microformat in 96 well design (Ibidi, Inc). PCOs were treated with physiologic FOLFIRINOX or gemcitabine + nab-paclitaxel (nab-pac) including single agent controls. Response was assayed by growth using timecourse data from 0h to 72h using high content imaging (Cytation 5). At 72h, the PCOs were stained using four distinct CSC markers (CD44, CD133, CD117, and EpCAM). Images were aligned using thresholded root mean standard deviation of 75μm and tracked for normalized change in longest diameter. At 72h, CSC markers were tracked using % area positive from brightfield region of interest. Response was tracked by effect size Glass’s Delta (GΔ) defined as the difference between the mean of media control and treatment group normalized to standard deviation of media control. Results: PCOs maintained nuclear atypia and high nuclear-chromatin ratios consistent with matured xenografts. AMPC1 showed similar sensitivity with growth arrest by PCO Δ diameter for gem + nab-pac (mean = +2.2%, GΔ = 1.29) and FOLFIRINOX (mean = 0.0%, GΔ = 1.33). AMPC1 had CSC enrichment for CD44 after treatment with nab-pac (GΔ = 0.93), CD133 expression after treatment with gem + nab-pac (GΔ = 0.75), CD117 after treatment with nab-pac treatment (GΔ = 0.80), and EpCAM after treatment with gem + nab-pac (GΔ = 0.78). In AMPC1, interquartile analysis revealed increased CSC expression conferred reduced growth for CD133 (+72% v. 37%, p<0.005) and CD117 (+81% v. +25%, p<0.005). Normalized Δ diameter across PDAC PCOs revealed PDAC1 had reduced sensitivity to gem + nab-pac (mean = +16.0%, GΔ = 1.2) vs. PDAC2 (Mean = 2.1%, GΔ = 1.65). PDAC1 had CSC enrichment with increased % area of CD44+ after treatment with gem + nab-pac (GΔ = 1.13). In PDAC2, interquartile analysis revealed increased CSC expression conferred decreased growth rate for CD133 (+65% v. +36%, p< 0.005) and CD117 (+75% v. +38%, p<0.005). Conclusions: PDAC and AMPC PCOs reveal oligoclonal expression of CSC markers within a population. Increased CSC expression for CD133 and CD117 within a population consistently revealed decreased growth within an organoid population. CSC expression (CD44, CD133, CD117, and EpCAM) was found to be enriched under therapeutic challenge with gem and nab-pac containing chemotherapy. This work supports the oligoclonal and dynamic nature of CSCs for pancreaticobiliary cancers in response to therapeutic challenge. Citation Format: Eleanor Riedl, Austin Stram, MD Shahadat Hossan, Ethan S. Lin, Luke Koeppel, Jamie Warner, Sam Lubner, Stephanie McGregor, Wei Zhang, Jeremy Kratz. Tracking cancer stem cell expression of pancreatic and ampullary cancer organoids in response to chemotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5809.

Biophysical cues of in vitro biomaterials-based artificial extracellular matrix guide cancer cell plasticity.

Clinical evidence supports a role for the extracellular matrix (ECM) in cancer plasticity across multiple tumor types. The lack of in vitro models that represent the native ECMs is a significant challenge for cancer research and drug discovery. Therefore, a major motivation for developing new tumor models is to create the artificial ECM in vitro. Engineered biomaterials can closely mimic the architectural and mechanical properties of ECM to investigate their specific effects on cancer progression, offering an alternative to animal models for the testing of cancer cell behaviors. In this review, we focused on the biomaterials from different sources applied in the fabrication of the artificial ECM and their biophysical cues to recapitulate key features of tumor niche. Furthermore, we summarized how the distinct biophysical cues guided cell behaviors of cancer plasticity, including morphology, epithelial-to-mesenchymal transition (EMT), enrichment of cancer stem cells (CSCs), proliferation, migration/invasion and drug resistance. We also discuss the future opportunities in using the artificial ECM for applications of tumorigenesis research and precision medicine, as well as provide useful messages of principles for designing suitable biomaterial scaffolds.

Epithelial-mesenchymal transition in cancer stem cells: Therapeutic implications.

Cancer stem cells (CSCs) are cancer cells that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. CSCs may generate tumors through the processes of self-renewal and differentiation into multiple cell types. CSCs present in tumors are normally resistant to conventional therapy and may contribute to tumor recurrence. Tumor residuals present after therapy, with CSCs enrichment, have all the hallmarks of epithelial-mesenchymal transition (EMT). In this review, we discuss the relationship between EMT and CSCs in cancer progression and its therapeutic implications in oral squamous cell carcinoma.

Canonical Wnt Pathway Is Involved in Chemoresistance and Cell Cycle Arrest Induction in Colon Cancer Cell Line Spheroids

The presence of cancer stem cells (CSCs) has been associated with the induction of drug resistance and disease recurrence after therapy. 5-Fluorouracil (5FU) is widely used as the first-line treatment of colorectal cancer (CRC). However, its effectiveness may be limited by the induction of drug resistance in tumor cells. The Wnt pathway plays a key role in the development and CRC progression, but it is not clearly established how it is involved in CSCs resistance to treatment. This work aimed to investigate the role played by the canonical Wnt/β-catenin pathway in CSCs resistance to 5FU treatment. Using tumor spheroids as a model of CSCs enrichment of CRC cell lines with different Wnt/β-catenin contexts, we found that 5FU induces in all CRC spheroids tested cell death, DNA damage, and quiescence, but in different proportions for each one: RKO spheroids were very sensitive to 5FU, while SW480 were less susceptible, and the SW620 spheroids, the metastatic derivative of SW480 cells, displayed the highest resistance to death, high clonogenic capacity, and the highest ability for regrowth after 5FU treatment. Activating the canonical Wnt pathway with Wnt3a in RKO spheroids decreased the 5FU-induced cell death. But the Wnt/β-catenin pathway inhibition with Adavivint alone or in combination with 5FU in spheroids with aberrant activation of this pathway produced a severe cytostatic effect compromising their clonogenic capacity and diminishing the stem cell markers expression. Remarkably, this combined treatment also induced the survival of a small cell subpopulation that could exit the arrest, recover SOX2 levels, and re-grow after treatment.

Surface roughness modulates EGFR signaling and stemness of triple-negative breast cancer cells.

Introduction: Cancer stem cells (CSC), a major culprit of drug-resistant phenotypes and tumor relapse, represent less than 2 % of the bulk of TNBC cells, making them difficult to isolate, study, and thus, limiting our understanding of the pathogenesis of the disease. Current methods for CSC enrichment, such as 3D spheroid culture, genetic modification, and stem cell conditioning, are time consuming, expensive, and unsuitable for high-throughput assays. One way to address these limitations is to use topographical stimuli to enhance CSC populations in planar culture. Physical cues in the breast tumor microenvironment can influence cell behavior through changes in the mechanical properties of the extracellular matrix (ECM). In this study, we used topographical cues on polystyrene films to investigate their effect on the proteome and stemness of standard TNBC cell lines. Methods: The topographical polystyrene-based array was generated using razor printing and polishing methods. Proteome data were analyzed and enriched bioprocesses were identified using R software. Stemness was assessed measuring CD44, CD24 and ALDH markers using flow cytometry, immunofluorescence, detection assays, and further validated with mammosphere assay. EGF/EGFR expression and activity was evaluated using enzyme-linked immunosorbent assay (ELISA), immunofluorescence and antibody membrane array. A dose-response assay was performed to further investigate the effect of surface topography on the sensitivity of cells to the EGFR inhibitor. Results: Surface roughness enriched the CSC population and modulated epidermal growth factor receptor (EGFR) signaling activity in TNBC cells. Enhanced proliferation of MDA-MB-468 cells in roughness correlated with upregulation of the epidermal growth factor (EGF) ligand, which in turn corresponded with a 3-fold increase in the expression of EGFR and a 42% increase in its phosphorylation compared to standard smooth culture surfaces. The results also demonstrated that phenotypic changes associated with topographical (roughness) stimuli significantly decreased the drug sensitivity to the EGFR inhibitor gefitinib. In addition, the proportion of CD44+/CD24-/ALDH+ was enhanced on surface roughness in both MDA-MB-231 and MDA-MB-468 cell lines. We also demonstrated that YAP/TAZ activation decreased in a roughness-dependent manner, confirming the mechanosensing effect of the topographies on the oncogenic activity of the cells. Discussion: Overall, this study demonstrates the potential of surface roughness as a culture strategy to influence oncogenic activity in TNBC cells and enrich CSC populations in planar cultures. Such a culture strategy may benefit high-throughput screening studies seeking to identify compounds with broader tumor efficacy.

A153 PYCRL LACTYLATION AS A POTENTIAL REGULATOR OF CANCER STEM CELL METABOLISM IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA (ESCC)

Abstract Background Patients with esophageal malignancy have a 5-year survival rate of only 14% in Canada. This high mortality rate is due to three factors: late diagnosis, difficulty in surgically removing the tumor because of its localization, and treatment resistance. Resistance can be developed after prolonged exposure to anti-cancer drugs and/or radiation. Indeed 30% of patients will not respond to treatment or will relapse. Resistance has been mainly ascribed to the presence of cancer stem cells (CSCs) inside the tumor. However, no treatment specifically directed against CSCs is available to patients. Purpose Thus, targeting CSCs is a promising strategy to improve the survival of patients with esophageal squamous cell carcinoma (ESCC), the most common type of esophageal cancer worldwide. Therefore, a better understanding of the molecular mechanisms occurring during long-term exposure to cancer treatments is imperative. Method Herein, we developed an unbiased approach to identify new players in chemotherapy and radiotherapy resistance development in ESCC. We established radio- (R), chemo- (C), and radiochemo-resistant (RC) human ESCC cell lines using prolonged exposure to radiation and/or chemotherapeutic agent 5-FU, respectively. Result(s) The enrichment in CSCs in all treated cell lines was demonstrated by an increase of ALDH1high cells and CD24high/CD44high cells in flow cytometry. We then used a proteomic approach to identify new players in treatment resistance. Interestingly, pathway analysis pointed out to alterations in energy metabolism as well as amino acid metabolism. Seahorse assays showed that resistant cell lines have a lower respiration rate than control cells, while glycolysis remains unchanged. To further characterize these metabolic changes, we performed an unbiased metabolomic study and confirmed a decrease in amino acid levels such as proline, in resistant cell lines. Recently, metabolic regulation has been linked to a new post-translational modification, lactylation. Proteomic data were re-analyzed looking for lactylated protein and found, amongst others, PYCRL, an enzyme implicated in proline biosynthesis, as one of the most differentially lactylated proteins in treated cell lines compared to control. PYCRL lactylation was confirmed using immuno-fluorescence colocalization and immuno-precipitation. Lastly, using AlphaFold, preliminary results point toward the importance of PYCRL lactylation impairing PYCRL homomultimerization. Conclusion(s) To conclude, our results suggest an important role of proline metabolism following long-term treatment in ESCC. This study is a first step toward the identification of new targets to fight treatment resistance in ESCC patients. Please acknowledge all funding agencies by checking the applicable boxes below CAG, CIHR, Other Please indicate your source of funding; FRQ Disclosure of Interest None Declared

Venetoclax-Resistant T-ALL Cells Display Distinct Cancer Stem Cell Signatures and Enrichment of Cytokine Signaling

Therapy resistance remains one of the major challenges for cancer treatment that largely limits treatment benefits and patient survival. The underlying mechanisms that lead to therapy resistance are highly complicated because of the specificity to the cancer subtype and therapy. The expression of the anti-apoptotic protein BCL2 has been shown to be deregulated in T-cell acute lymphoblastic leukemia (T-ALL), where different T-ALL cells display a differential response to the BCL2-specific inhibitor venetoclax. In this study, we observed that the expression of anti-apoptotic BCL2 family genes, such as BCL2, BCL2L1, and MCL1, is highly varied in T-ALL patients, and inhibitors targeting proteins coded by these genes display differential responses in T-ALL cell lines. Three T-ALL cell lines (ALL-SIL, MOLT-16, and LOUCY) were highly sensitive to BCL2 inhibition within a panel of cell lines tested. These cell lines displayed differential BCL2 and BCL2L1 expression. Prolonged exposure to venetoclax led to the development of resistance to it in all three sensitive cell lines. To understand how cells developed venetoclax resistance, we monitored the expression of BCL2, BCL2L1, and MCL1 over the treatment period and compared gene expression between resistant cells and parental sensitive cells. We observed a different trend of regulation in terms of BCL2 family gene expression and global gene expression profile including genes reported to be expressed in cancer stem cells. Gene set enrichment analysis (GSEA) showed enrichment of cytokine signaling in all three cell lines which was supported by the phospho-kinase array where STAT5 phosphorylation was found to be elevated in resistant cells. Collectively, our data suggest that venetoclax resistance can be mediated through the enrichment of distinct gene signatures and cytokine signaling pathways.

AT7867 Inhibits the Growth of Colorectal Cancer Stem-Like Cells and Stemness by Regulating the Stem Cell Maintenance Factor Ascl2 and Akt Signaling.

Cancer stem cells (CSCs) are the core factors leading to recurrence, insensitivity to radiotherapy and chemotherapy, and immunotherapy resistance in patients with colorectal cancer. AT7867, a potent oral AKT inhibitor, was found to have antitumor activity in colorectal cancer; however, the effect on colorectal cancer stem cells is still unclear. This study was conducted to clarify the molecular mechanism underlying the CSC growth inhibitory effects of AT7867. We cultured colorectal cancer cells (CRCs) in a serum-free medium and enriched colorectal cancer stem cells. Subsequently, the effects of AT7867 on CSCs were analyzed by CCK-8, colony formation, flow cytometry, and immunofluorescence assays. The results indicated that AT7867 induces G2/M phase arrest and cell apoptosis in cancer stem cells. Subsequently, we identified Ascl2 as the main gene affecting the stemness of colorectal cancer in AT7867 by RNA sequencing. The current study showed that Ascl2 is involved in the metastasis, invasion, and proliferation of CRCs. The next experiments demonstrated that overexpression of Ascl2 did affect the therapeutic effect of AT7867 on CRC stemness. Furthermore, compared with other Akt inhibitors, AT7867 could promote the differentiation of colorectal cancer stem cells. Thus, AT7867 might be a potential antitumor drug candidate to treat CRC by targeting CSCs.

Periostin secreted from podoplanin-positive cancer-associated fibroblasts promotes metastasis of gastric cancer by regulating cancer stem cells via AKT and YAP signaling pathway.

Cancer-associated fibroblasts (CAFs) are heterogeneous stromal cells present in the tumor microenvironment (TME), which play a critical role in gastric cancer (GC) progression. Here, we examined a subset of CAFs with high podoplanin (PDPN) expression, which is correlated with tumor metastasis and poor survival in GC patients. Animal models of gastric cancer liver metastasis monitored by micro-PET/CT confirmed that periostin (POSTN) derived from PDPN(+) CAFs regulated CAFs' pro-migratory ability. Mechanistically, PDPN(+) CAFs secreted POSTN to modulate cancer stem cells (CSCs) through FAK/AKT phosphorylation. Furthermore, POSTN could also activate FAK/YAP signaling in GC cells to produce increased amounts of IL-6, which in turn induced phosphorylation of PI3K/AKT in PDPN(+) CAFs. Prolonged PI3K/AKT pathway activation in PDPN(+) CAFs maintains the production of POSTN and the effect on CSC enrichment and GC cell migration. In conclusion, our study demonstrated a positive feedback loop between PDPN(+) CAFs and CSCs during GC progression and suggested a selective target for GC treatment.

A reliable mouse model of liver and lung metastasis by injecting esophageal cancer stem cells (CSCs) through tail-vein injection.

Esophageal Squamous Cell Carcinoma (ESCC) is a highly aggressive tumor with increased metastatic potential. Recent evidence suggests that esophageal CSCs have a crucial role in tumor initiation, progression, and resistance to conventional anti-cancer therapies. The study aimed to develop mouse model to mimic the late steps of the metastasis process using a tail-vein injection of esophageal CSCs. The sphere formation assay was used to enrich CSCs. For analysis of tumorigenicity, YM-1 adherent cells and enriched CSCs were injected subcutaneously into dorsal flank of nude mice. The expression of SLUG, E-cad, and CTHRC1 genes was examined by Real-Time qRT-PCR and immunohistochemistry (IHC) methods. To assess the metastatic potential of adherent YM-1 cells and their enriched CSCs, we injected the cells into the tail vein of nude mice. Our findings showed the up-regulation of SLUG and down-regulation of E-cad in the esophageal CSC-derived tumors (ECSCTs) compared to adherent cells-derived tumors. There was no statistically significant difference between CTHRC1 gene expressions in both groups of tumors. IHC staining confirmed the higher expression of SLUG protein in ECSCTs compared to adherent cell-derived tumors. Enriched CSCs were able to metastasize to the lungs and livers after three months, but, metastasis of adherent cells wasn't observed. Our study showed esophageal CSCs injected through the tail-vein injection can migrate and metastasize to the lung and liver after three months. The developed metastatic mouse model can be a valuable and relevant model to investigate the molecular and cellular mechanisms of metastasis and develop successful targeted therapies against ESCC. The present study is one of the few studies that investigate the metastasis of esophageal cancer stem cells (ESCC type) through injection into the tail vein of nude mice.

Suspension culture strategies to enrich colon cancer stem cells.

How to efficiently obtain high-purity cancer stem cells (CSCs) has been the basis of CSC research, but the optimal conditions for serum-free suspension culture of CSCs are still unclear. The present study aimed to define the optimal culture medium composition and culture time for the enrichment of colon CSCs via suspension culture. Suspension cell cultures of colon cancer DLD-1 cells were prepared using serum-free medium (SFM) containing variable concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to produce spheroids. Culture times were set at 10, 20 and 30 days. A total of nine different concentrations of EGF and bFGF were added to SFM to generate nine experimental groups. The proportions of CD44+, CD133+, and CD44+CD133+ double-positive spheroid cells were detected via flow cytometry. mRNA expression of stemness-, epithelial-mesenchymal transition- and Wnt/β-catenin pathway-associated genes was determined via reverse transcription-quantitative PCR. Self-renewal ability was evaluated by a sphere-forming assay. Tumorigenesis was studied in vitro using a colony formation assay and in vivo via subcutaneous cell injection in nude mice. It was found that the highest expression proportions of CD133+ and CD44+ spheroid cells were observed in group (G)9 (20 ng/ml EGF + 20 ng/ml bFGF) at 30 days (F=123.554 and 99.528, respectively, P<0.001), CD133+CD44+ cells were also observed in G9 at 30 days (and at 10 days in G3 and 20 days in G6; F=57.897, P<0.001). G9 at 30 days also displayed the highest expression of Krüppel-like factor 4, leucine-rich repeat-containing G protein-coupled receptor 5, CD44, CD133, Vimentin and Wnt-3a (F=22.682, 25.401, 3.272, 7.852, 13.331 and 17.445, respectively, P<0.001) and the lowest expression of E-cadherin (F=10.851, P<0.001). G9 at 30 days produced the highest yield of cell spheroids, as determined by a sphere forming assay (F=19.147, P<0.001); colony formation assays also exhibited the greatest number of colonies derived from G9 spheroids at 30 days (F=60.767, P<0.01), which also generated the largest mean tumor volume in the subcutaneous tumorigenesis xenograft model (F=12.539, P<0.01). In conclusion, 20 ng/ml EGF + 20 ng/ml bFGF effectively enriched colon CSCs when added to suspension culture for 30 days, and conferred the highest efficiency compared with other combinations.