Higher Clonogenicity Research Articles

Abstract 122: Fusobacterium nucleatum in saliva of oral cancer patients induces tumor stemness defense phenotype in cancer stem cells

Abstract Background: Oral squamous cell carcinoma (OSCC) is a highly aggressive cancer. Cancer Stem Cells (CSCs) having cell surface markers CD44, ALDH, and EpCAM+ have been identified in OSCC. However, the host pathogen interaction between CSCs and oral microbiome has not yet been studied. A number of studies have recently demonstrated the presence of specific oral bacteria populations and their lipopolysaccharides (LPS) in the tumor microenvironment (1). We previously reported pathogenic bacterial internalization in the CSCs (2). Based on the findings, we have developed an in-vitro model to investigate how oral microbiota may integrate into the tumor microenvironment's CSC population and control its activity. Here, we investigated the host/pathogen interaction between CSCs and FN. Methods: Oral bacteria FN was selectively isolated using FEA agar plates from oral saliva of oral cancer patients (n=10) at stage 3 or 4. The isolated bacteria was co cultured with SCC 25 cells at MOI of 1:50. After co culturing the stemness genes such as SOX 2, OCT 4, Nanog,LPS and ABCG2+ cancer stem cell markers were analysed. ABCG2+ CSCs and ABCG2- cells were immunomagnetically sorted and lysed followed by RNA extraction and RT QPCR was performed to observe if FN has selectively internalised in the CSCs. Also ABCG2+ and ABCG 2- cells were used to perform clonogenic assay. Results: Coculture of FN with SCC 25 cell line induced high expression of stemness genes SOX, OCT 4 and Nanog along with CSC markers ABCG2+. Moreover high expression of LPS was observed indicating the presence of FN in the coculture. Importantly live bacteria was found internalized in ABCG2+ cells but not in ABCG2- cells. High clonogenicity was observed in ABCG2+ CSCs compared to ABCG2- cells. Notably, we found that live bacteria and their LPS, mostly Fusobacterium nucleatum isolated (FN) from clinical subjects, were capable of invading CSCs in the in-vitro setting. Post the host-pathogen interaction; it enabled the activation of a niche modulatory tumor stemness defense (TSD) phenotype in the CSCs. These aggressive CSCs with the TSD phenotype have been found to have a critical role in the progression and relapse of oral cancer. Conclusion: Expression of stemness genes infers the niche defence mechanism of the CSCs of the tumour microenvironment due to infection of FN. Importantly presence of live bacteria in ABCG2+ CSCs indicates that FN selectively infects ABCG2+ CSCs but not ABCG2- cells. High clonogenicity of ABCG2+ CSCs confirms the TSD phenotypic attributes of the CSCs.

Chromosome 9p Duplication Promotes T-Cell Exhaustion and Enhances Stem Cell Clonogenic Potential in JAK2-Mutant Myeloproliferative Neoplasms

Myeloproliferative Neoplasms (MPNs) are a diverse group of clonal hematopoietic disorders originating from a single hematopoietic stem cell, causing an excessive production of mature blood cells. Classic forms of MPNs include polycythemia vera, essential thrombocythemia, and myelofibrosis. The most common mutation is a gain-of-function point mutation in the JAK2 gene, known as JAK2V617F. This mutation leads to constant activation of the JAK-STAT signaling pathway, resulting in the overgrowth of MPN cells. Furthermore, the variant allele frequency (VAF) of JAK2V617F has a significant impact on the severity and phenotype of the disease. Alongside many described genetic mutations, cytogenetic abnormalities are commonly observed in MPNs, particularly involving chromosome 9. As the JAK2 gene is located on the short arm of this chromosome, we hypothesized that chromosome 9 copy number abnormalities might be a disease modifier in JAK2V617F-mutant MPN patients. To characterize the biological effects of chromosome 9 copy number abnormalities on JAK2-mutated MPN cells, we analyzed circulating CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as monocytes and granulocytes from 32 MPN patients. Through Next Generation Sequencing, we categorized the patients into three main groups based on JAK2 mutation and copy number status: patients with two copies of the JAK2 gene with either heterozygous (VAF between 20% and 60%; n=10) or homozygous (VAF > 60% n=10) JAK2V617F mutation, or patients carrying mutant JAK2 and gene amplification (n=12). In-depth analysis of JAK2-amplified patients revealed that the amplification involved the entire chromosome 9p, thus including other gene loci like CD274, which encodes programmed death-ligand 1 (PD-L1). Further investigation of the order-of-events and clonal hierarchies through droplet digital PCR on CD34+ cell-derived colonies showed that most colonies of 9p-duplicated patients had three copies of JAK2, with 2 out of 3 alleles harboring the JAK2 mutation and that point mutations are frequently the initial pathogenic event in clonal evolution, followed by amplification of the JAK2-mutated allele. Functionally, CD34+ cells from +9p patients displayed high clonogenicity and gave rise to a greater number of primitive colonies (Colony Forming Unit-Granulocyte, Erythrocyte, Monocyte, Megakaryocyte, CFU-GEMM). As JAK2 hyperactivation had been previously reported to lead to increased PD-L1 expression, we further explored the functional significance of CD274 amplification in +9p patients. Our analysis showed increased PD-L1 messenger RNA and protein levels in +9p patient CD14+ monocytes compared to those from patients carrying only two copies of chromosome 9. Moreover, immunofluorescence analysis demonstrated significant re-localization of PD-L1 to the cytoplasmic membrane in monocytes from +9p patients, but not in JAK2V617F-homozygous patients (panel A). Increased levels of PD-L1, an immune checkpoint known to curb T cell activation, led us to analyze the T cell compartment, which resulted enriched in CD3+/CD8+/CD57-/PD-1+ exhausted T-cells in +9p patients compared to other MPN patients and healthy donors (panel B). In conclusion, our comprehensive characterization of the molecular interplay between JAK2V617F and chromosome 9 alterations, along with their immunological implications due to PD-L1 hyperactivation, fills a critical knowledge gap and provides valuable insights into the disease progression of 9p-MPNs. Further analysis is ongoing to explore the associations between 9p duplication and hematological parameters in 9p-MPN patients for a better understanding of the clinical implications of this genetic abnormality in MPNs.

Stabilization of hESCs in two distinct substates along the continuum of pluripotency.

A detailed understanding of the developmental substates of human pluripotent stem cells (hPSCs) is needed to optimize their use in cell therapy and for modeling early development. Genetic instability and risk of tumorigenicity of primed hPSCs are well documented, but a systematic isogenic comparison between substates has not been performed. We derived four hESC lines in naive human stem cell medium (NHSM) and generated isogenic pairs of NHSM and primed cultures. Through phenotypic, transcriptomic, and methylation profiling, we identified changes that arose during the transition to a primed substate. Although early NHSM cultures displayed naive characteristics, including greater proliferation and clonogenic potential compared with primed cultures, they drifted toward a more primed-like substate over time, including accumulation of genetic abnormalities. Overall, we show that transcriptomic and epigenomic profiling can be used to place human pluripotent cultures along a developmental continuum and may inform their utility for clinical and research applications.

Abstract 355: Molecular characterization of BRAF V600E CRC in response to Src-targeted therapy

Abstract Oncogenic mutations in B-type Raf Kinase (BRAF) (particularly V600E) are present in 7-10% of metastatic colorectal cancer (mCRC), conferring resistance to conventional therapies and a poor prognosis. Src Family Kinases (SFKs) play a relevant role in CRC, where Src activation is associated with an aggressive phenotype and a worse prognosis; and could be involved in chemoresistance mechanisms. Dasatinib is a potent Src inhibitor with demonstrated anti-proliferative activity, although deeper knowledge is needed. Thus, we aimed to explore the role of Src in BRAF V600E CRC and to investigate this tumor subgroup in response to Dasatinib. SRC activation was evaluated by Western Blot. We generated Knock-Out (KO) Src cell lines by CRISPR/Cas9 system and stable Src overexpression (OE) by lentivirus. Cell viability was analysed by crystal violet and MTS assay. Phospho-kinase proteins screening was performed using the Proteome Profiler Human Phospho-Kinase Array kit. Expression levels of Src and phospho-Src (p-Src) displayed an inverse correlation with IC50 values for Dasatinib in a panel of 7 CRC cell lines including BRAF V600E (HT29, COLO201, RKO, LS411N) and BRAF WT (DIFI, HCA7 and LIM1215), and these doses were able to inhibit Src activation in all cell lines. Focusing on HT29 cells, inhibition of p-Src occurs in a dose-dependent manner and in shortened periods of treatment. Dasatinib showed a significant inhibition of proliferation and migration on HT29. Moreover, stable Src-OE HT29 cells showed higher clonogenicity, migration and proliferation capacities, as well as higher resistance to Dasatinib. Conversely, HT29 Src KO cells were more sensitive. In line with those results, silencing Src in other BRAF V600E cells (RKO and LS411N) also resulted in a remarkable reduction in clonogenicity and migration abilities. Xenograft mouse model of SRC depletion showed that the growth of SRC KO tumors was significantly slower than WT tumors. Phospho-kinase array quantification in basal and Src-OE HT29 cells showed up- and downregulated proteins related to classical tumorigenic signaling pathways such as MAPK (JNK1/2, MSK), PI3K/Akt (Akt, TOR, GSK3), and others. Further studies to explore resistance mechanisms mediated by Src/SFKs are required. To conclude, lower p-Src expression at baseline is related to a higher resistance to Dasatinib in all CRC cell lines. Dasatinib showed a significant anti-proliferative activity in a subset of CRC cell lines and regulates the activity of Src in a time and dose-dependent manner. Modified expression of Src (OE and KO) in BRAF V600E CRC cells significantly regulates clonogenicity, proliferation and migration abilities in vitro. Src overexpression is responsible of the acquired resistance to Dasatinib and regulates several pathways that could be involved in the tumorigenesis of BRAF V600E CRC, although further studies to explore resistance mechanisms mediated by Src/SFKs are required. Citation Format: Beatriz Rubio Cuesta, Beatriz Gil Calderón, Patricia LLamas Granda, Jacinto Sarmentero, Carlos Carretero Puche, María del Carmen Riesco, Rocío García Carbonero, Beatriz Soldevilla. Molecular characterization of BRAF V600E CRC in response to Src-targeted therapy [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 355.

Portal fibroblasts with mesenchymal stem cell features form a reservoir of proliferative myofibroblasts in liver fibrosis.

In liver fibrosis, myofibroblasts derive from HSCs and as yet undefined mesenchymal cells. We aimed to identify portal mesenchymal progenitors of myofibroblasts. Portal mesenchymal cells were isolated from mouse bilio-vascular tree and analyzed by single-cell RNA-sequencing. Thereby, we uncovered the landscape of portal mesenchymal cells in homeostatic mouse liver. Trajectory analysis enabled inferring a small cell population further defined by surface markers used to isolate it. This population consisted of portal fibroblasts with mesenchymal stem cell features (PMSCs), i.e., high clonogenicity and trilineage differentiation potential, that generated proliferative myofibroblasts, contrasting with nonproliferative HSC-derived myofibroblasts (-MF). Using bulk RNA-sequencing, we built oligogene signatures of the two cell populations that remained discriminant across myofibroblastic differentiation. SLIT2, a prototypical gene of PMSC/PMSC-MF signature, mediated profibrotic and angiogenic effects of these cells, which conditioned medium promoted HSC survival and endothelial cell tubulogenesis. Using PMSC/PMSC-MF 7-gene signature and slit guidance ligand 2 fluorescent in situ hybridization, we showed that PMSCs display a perivascular portal distribution in homeostatic liver and largely expand with fibrosis progression, contributing to the myofibroblast populations that form fibrotic septa, preferentially along neovessels, in murine and human liver disorders, irrespective of etiology. We also unraveled a 6-gene expression signature of HSCs/HSC-MFs that did not vary in these disorders, consistent with their low proliferation rate. PMSCs form a small reservoir of expansive myofibroblasts, which, in interaction with neovessels and HSC-MFs that mainly arise through differentiation from a preexisting pool, underlie the formation of fibrotic septa in all types of liver diseases.

Oral stem cells, decoding and mapping the resident cells populations.

ABSTRACTThe teeth and their supporting tissues provide an easily accessible source of oral stem cells. These different stem cell populations have been extensively studied for their properties, such as high plasticity and clonogenicity, expressing stem cell markers and potency for multilineage differentiation in vitro. Such cells with stem cell properties have been derived and characterised from the dental pulp tissue, the apical papilla region of roots in development, as well as the supporting tissue of periodontal ligament that anchors the tooth within the alveolar socket and the soft gingival tissue. Studying the dental pulp stem cell populations in a continuously growing mouse incisor model, as a traceable in vivo model, enables the researchers to study the properties, origin and behaviour of mesenchymal stem cells. On the other side, the oral mucosa with its remarkable scarless wound healing phenotype, offers a model to study a well-coordinated system of healing because of coordinated actions between epithelial, mesenchymal and immune cells populations. Although described as homogeneous cell populations following their in vitro expansion, the increasing application of approaches that allow tracing of individual cells over time, along with single-cell RNA-sequencing, reveal that different oral stem cells are indeed diverse populations and there is a highly organised map of cell populations according to their location in resident tissues, elucidating diverse stem cell niches within the oral tissues. This review covers the current knowledge of diverse oral stem cells, focusing on the new approaches in studying these cells. These approaches “decode” and “map” the resident cells populations of diverse oral tissues and contribute to a better understanding of the “stem cells niche architecture and interactions. Considering the high accessibility and simplicity in obtaining these diverse stem cells, the new findings offer potential in development of translational tissue engineering approaches and innovative therapeutic solutions.

Human skeletal muscle CD90+ fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients

Type 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle function and degeneration of the skeletal muscles. However, the mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscle of T2DM patients exhibit degenerative remodeling of the extracellular matrix that is associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90)-the FAPCD90+. We identify platelet-derived growth factor (PDGF) as a key FAP regulator, as it promotes proliferation and collagen production at the expense of adipogenesis. FAPsCD90+ display a PDGF-mimetic phenotype, with high proliferative activity, clonogenicity, and production of extracellular matrix. FAPCD90+ proliferation was reduced by invitro treatment with metformin. Furthermore, metformin treatment reduced FAP content in T2DM patients. These data identify a PDGF-driven conversion of a subpopulation of FAPs as a key event in the fibrosis development in T2DM muscle.

Marker-free lineage tracing reveals an environment-instructed clonogenic hierarchy in pancreatic cancer.

Effective treatments for pancreatic ductal adenocarcinoma (PDAC) are lacking, and targeted agents have demonstrated limited efficacy. It has been speculated that a rare population of cancer stem cells (CSCs) drives growth, therapy resistance, and rapid metastatic progression in PDAC. These CSCs demonstrate high clonogenicity invitro and tumorigenic potential invivo. However, their relevance in established PDAC tissue has not been determined. Here, we use marker-independent stochastic clonal labeling, combined with quantitative modeling of tumor expansion, to uncover PDAC tissue growth dynamics. We find that in contrast to the CSC model, all PDAC cells display clonogenic potential in situ. Furthermore, the proximity to activated cancer-associated fibroblasts determines tumor cell clonogenicity. This means that the microenvironment is dominant in defining the clonogenic activity of PDAC cells. Indeed, manipulating the stroma by Hedgehog pathway inhibition alters the tumor growth mode, revealing that tumor-stroma crosstalk shapes tumor growth dynamics and clonal architecture.

Marker-Free Lineage Tracing Reveals an Environment-Instructed Clonogenic Hierarchy in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies. Effective treatment options are currently lacking, and targeted agents have so far demonstrated limited efficacy. It has been speculated that a rare population of cancer stem cells (CSCs) drives growth, therapy resistance, and rapid metastatic progression in PDAC. These CSCs are characterized by expression of genes associated with immature cells, and demonstrate high clonogenicity in vitro and tumorigenic potential in vivo. However, the unique relevance of CSCs in established PDAC tissue has not been determined. Here, we used a marker-independent stochastic clonal labeling system, in combination with a quantitative model of tumor expansion, to uncover the growth dynamics of PDAC tissue. We found that in situ all PDAC cells display clonogenic potential, which is in stark contrast to a strict hierarchical CSC model. Furthermore, we uncover that the proximity to activated cancer-associated fibroblasts determines which tumor cells actively drive tumor expansion. This means that the microenvironment is a dominant actor in defining the clonogenic activity of PDAC cells. Indeed, manipulating the PDAC associated stroma by Hedgehog pathway inhibition, altered the mode of tumor growth. These results reveal how tumor-stroma crosstalk shapes the growth dynamics and clonal architecture of these malignancies, and how perturbance of the stroma impacts on important aspects of PDAC biology.

Abstract A80: Targeting ovarian cancer stem cell by Dot1L inhibition

Abstract Cancer stem cells (CSCs) are a specific population of cancer cells that are responsible for tumor initiation, chemoresistance, and metastasis. Previously, our lab has demonstrated ovarian cancer cells with high aldehyde dehydrogenase (ALDH) activities have multiple stem cell-like characteristics, including self-renewal, high clonogenicity, and chemoresistance. Here we identify that H3K79 methyltransferase DOT1L (disruptor of telomeric silencing-1-like) is a critical factor in self-renewal and tumor initiation capabilities of ovarian cancer stem cells (OCSCs). Firstly, we found that DOT1L have higher expression in ALDH(+)/CD133(+) OCSCs compared to double negative cells. Ovarian cancer cells with DOT1L-knockdown have reduced CSC fractions in flow cytometry analysis. Besides, knockdown of DOT1L expression impairs clonogenicity characteristics of CSCs, according to in vitro colony-forming assay and spheroid formation assay results. In order to effectively inhibit the enzymatic activity of DOT1L, we applied specific DOT1L inhibitor (DOT1Li) EPZ5676 to specifically target OCSCs and confirmed that the treatment of DOT1Li was able to block H3K79 methylation in ovarian cancer cells, and effectively represses the expression of stemness-associated transcriptional factors. Results from RNA-seq analysis and gene set enrichment assay have indicated that multiple stem-related pathways are repressed in DOT1Li-treated OCSCs. Furthermore, DOT1Li treatment not only shows inhibitory effects on colony forming in vitro but also delays tumor initiation in vivo compared to control treatment, which validates the effects of DOT1Li on CSCs inhibition. Based on our result, we infer that DOT1L is critical for self-renewal and tumor initiation abilities in ovarian cancer stem cells, and DOT1L inhibition by specific inhibitor provides a potential therapeutic strategy to target CSCs in ovarian cancer treatment. Citation Format: Yaqi Zhang, Yinu Wang, Guangyuan Zhao, Daniela Matei. Targeting ovarian cancer stem cell by Dot1L inhibition [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A80.

Progenitor Cells Activated by Platelet Lysate in Human Articular Cartilage as a Tool for Future Cartilage Engineering and Reparative Strategies.

Regenerative strategies for human articular cartilage are still challenging despite the presence of resident progenitor cell population. Today, many efforts in the field of regenerative medicine focus on the use of platelet derivatives due to their ability to reactivate endogenous mechanisms supporting tissue repair. While their use in orthopedics continues, mechanisms of action and efficacy need further characterization. We describe that the platelet lysate (PL) is able to activate chondro-progenitor cells in a terminally differentiated cartilage tissue. Primary cultures of human articular chondrocytes (ACs) and cartilage explants were set up from donor hip joint biopsies and were treated in vitro with PL. PL recruited a chondro-progenitors (CPCs)-enriched population from ex vivo cartilage culture, that showed high proliferation rate, clonogenicity and nestin expression. CPCs were positive for in vitro tri-lineage differentiation and formed hyaline cartilage-like tissue in vivo without hypertrophic fate. Moreover, the secretory profile of CPCs was analyzed, together with their migratory capabilities. Some CPC-features were also induced in PL-treated ACs compared to fetal bovine serum (FBS)-control ACs. PL treatment of human articular cartilage activates a stem cell niche responsive to injury. These facts can improve the PL therapeutic efficacy in cartilage applications.

Bif-1/Endophilin B1/SH3GLB1 regulates bone homeostasis.

Skeletal tissue homeostasis is maintained via the balance of osteoclastic bone resorption and osteoblastic bone formation. Autophagy and apoptosis are essential for the maintenance of homeostasis and normal development in cells and tissues. We found that Bax-interacting factor 1 (Bif-1/Endophillin B1/SH3GLB1), involving in autophagy and apoptosis, was upregulated during osteoclastogenesis. Furthermore, mature osteoclasts expressed Bif-1 in the cytosol, particularly the perinuclear regions and podosome, suggesting that Bif-1 regulates osteoclastic bone resorption. Bif-1-deficient (Bif-1 -/- ) mice showed increased trabecular bone volume and trabecular number. Histological analyses indicated that the osteoclast numbers increased in Bif-1 -/- mice. Consistent with the in vivo results, osteoclastogenesis induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) was accelerated in Bif-1 -/- mice without affecting RANKL-induced activation of RANK downstream signals, such as NF-κB and mitogen-activated protein kinases (MAPKs), CD115/RANK expression in osteoclast precursors, osteoclastic bone-resorbing activity and the survival rate. Unexpectedly, both the bone formation rate and osteoblast surface substantially increased in Bif-1 -/- mice. Treatment with β-glycerophosphate (β-GP) and ascorbic acid (A.A) enhanced osteoblastic differentiation and mineralization in Bif-1 -/- mice. Finally, bone marrow cells from Bif-1 -/- mice showed a significantly higher colony-forming efficacy by the treatment with or without β-GP and A.A than cells from wild-type (WT) mice, suggesting that cells from Bif-1 -/- mice had higher clonogenicity and self-renewal activity than those from WT mice. In summary, Bif-1 might regulate bone homeostasis by controlling the differentiation and function of both osteoclasts and osteoblasts (235 words).

An Efficient Method for Cloning Gastrointestinal Stem Cells From Patients via Endoscopic Biopsies

An Efficient Method for Cloning Gastrointestinal Stem Cells From Patients via Endoscopic Biopsies

Circulating endothelial progenitors in vascular repair

Endothelial progenitor cells (EPCs) display pro-angiogenic properties that confer to them a therapeutic potential for treating ischemic lesions. In culture, EPCs generate Endothelial Colony Forming Cells (ECFCs) that have endothelial features but still retain properties of stem/progenitor cells. EPCs can be obtained either from cord blood (CB) or adult blood (AB). For clinical perspectives, both CB-ECFCs and AB-ECFCs have advantages and inconveniences. On one hand, CB-ECFCs are allogenic, with potential immune rejection, but are more clonogenic, proliferative and angiogenic than AB-ECFCs. We have found that these cells display high inter-individual variation in terms of clonogenic profile. Low clonogenic CB-ECFCs had functional properties similar to those of AB-ECFCs. Conversely, a high initial clonogenicity was associated to an enhanced proliferation, angiogenic potential and to an increased expression of genes linked to stem cell-ness. Selecting CB-ECFCs according the initial clonogenicity represent a relevant marker of their potential efficacy on vascular repair. On the other hand, AB-ECFCs are autologous, and should be immunologically well tolerated, but these cells give rise to only few colonies, proliferate less and are less angiogenic than CB-ECFCs. In order to go further in CB-/AB- characterization, we have analyzed the mechanisms of AB-ECFC premature senescence, and shown that GDF15 had pivotal role in this context. Taken together, these new data constitute important steps toward clinical use of EPCs.

Abstract 2437: Novel metabolic adaptations support proliferation of African American prostate cancer cells under hypoxia

Abstract Hypoxia in prostate cancer (PCa) selects for aggressive clones and causes treatment failure, metastatic progression and mortality. Despite the key role of hypoxia in prostate carcinogenesis, little is known about the specific effect of hypoxia in African Americans, a race that faces a serious PCa-related health disparity with the highest incidence and mortality rate. We hypothesize that metabolic adaptation to hypoxia is one mechanism that contributes to PCa aggressiveness in African Americans. Therefore, in the present work we focused on the metabolic adaptations, both in vitro and in vivo, contributing to proliferation of African American PCa cells under hypoxic conditions. PCa cells from men of African American ancestry (E006AA-hT, MDA PCa 2b, WFCB17 and 22Rv1) showed proliferation and significantly higher clonogenicity under hypoxia (1% O2) compared to normoxia. In contrast, Caucasian PCa cells (LNCaP and PC3) showed significant growth inhibition under similar hypoxic condition. The proliferation under hypoxia in African American PCa cells was associated with higher beta-oxidation of lipids as measured by malate response in Oroboros Oxygraph system. In addition, [18F]-fluoro-4-thia-oleate ([18F]-FTO) uptake in African American PCa cells under hypoxia was more than 2-fold higher (p <0.001) compared to normoxic cells, confirming higher beta-oxidation in hypoxic PCa cells. Furthermore, African American PCa cells showed more than 2-fold (p <0.001) higher uptake of [11C]-acetate under hypoxia, suggesting increased fatty acid synthesis during hypoxia in African American PCa cells. This suggests that African American PCa cells may increase fatty acid synthesis under hypoxia to provide metabolic fuel. Fatty acid oxidation is associated with mitochondrial metabolism; accordingly, we observed higher oxidative phosphorylation, complex II activity and expression under hypoxia in African American PCa cells. Importantly, treatment with complex II inhibitor Atpenin A5 (1-2 microM) strongly reduced the clonogenicity (51%-79%, p <0.0001) of African American PCa cells under hypoxia. In vivo, mPET/CT imaging using [18F]-FTO and [18F]-fluoromisonidazole as PET tracers showed greater tumor uptake of both tracers in 22Rv1 tumors in male athymic nude mice, suggesting that hypoxia and fatty acid oxidation are associated with African American PCa growth. Furthermore, analyses of 22Rv1 tumor tissues by confocal microscopy showed higher level of carnitine palmitoyltransferase 1 (a key regulator of β-oxidation) and succinate dehydrogenase B (a subunit of complex II) in hypoxic areas (pimonidazole+) of the tumor. Taken together, these data demonstrate that proliferation of African American PCa cells under hypoxia is supported by high lipid beta-oxidation, lipid synthesis and complex II activity, providing a novel mechanism to promote disease aggressiveness. Citation Format: Gati Panigrahi, Prakash Praharaj, Kiran Sai, Gargi Mahapatra, Taylor Peak, Sierra Patterson, Hakeem Oufkir, Anthony Molina, Steven Kridel, Gagan Deep. Novel metabolic adaptations support proliferation of African American prostate cancer cells under hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2437.

Hypericin affects cancer side populations via competitive inhibition of BCRP

ObjectiveCancer stem-like cells (CSLCs) are considered a root of tumorigenicity and resistance. However, their identification remains challenging. The use of the side population (SP) assay as a credible marker of CSLCs remains controversial. The SP assay relies on the elevated activity of ABC transporters that, in turn, can be modulated by hypericin (HYP), a photosensitizer and bioactive compound of St. John’s Wort (Hypericum perforatum), a popular over-the-counter antidepressant. Here we aimed to comprehensively characterize the SP phenotype of cancer cells and to determine the impact of HYP on these cells. MethodsFlow cytometry and sorting-based assays were employed, including CD24-, CD44-, CD133-, and ALDH-positivity, clonogenicity, 3D-forming ability, ABC transporter expression and activity, and intracellular accumulation of HYP/Hoechst 33342. The tumorigenic ability of SP, nonSP, and HYP-treated cells was verified by xenotransplantation into immunodeficient mice. ResultsThe SP phenotype was associated with elevated expression of several investigated transporters and more intensive growth in non-adherent conditions but not with higher clonogenicity, tumorigenicity or ALDH-positivity. Despite stimulated BCRP level and MRP1 activity, HYP reversibly decreased the SP proportion, presumably via competitive inhibition of BCRP. HYP-selected SP cells acquired additional traits of resistance and extensively eliminated HYP. ConclusionsOur results suggest that SP is not an unequivocal CSLC-marker. However, SP could play an important role in modulating HYP-treatment and serve as a negative predictive tool for HYP-based therapies. Moreover, the use of supplements containing HYP by cancer patients should be carefully considered, due to its proposed effect on drug efflux and complex impact on tumor cells, which have not yet been sufficiently characterized.

Isolation and characterization of olfactory ecto-mesenchymal stem cells from eight mammalian genera

BackgroundStem cell-based therapies are an attractive option to promote regeneration and repair defective tissues and organs. Thanks to their multipotency, high proliferation rate and the lack of major ethical limitations, “olfactory ecto-mesenchymal stem cells” (OE-MSCs) have been described as a promising candidate to treat a variety of damaged tissues. Easily accessible in the nasal cavity of most mammals, these cells are highly suitable for autologous cell-based therapies and do not face issues associated with other stem cells. However, their clinical use in humans and animals is limited due to a lack of preclinical studies on autologous transplantation and because no well-established methods currently exist to cultivate these cells. Here we evaluated the feasibility of collecting, purifying and amplifying OE-MSCs from different mammalian genera with the goal of promoting their interest in veterinary regenerative medicine.Biopsies of olfactory mucosa from eight mammalian genera (mouse, rat, rabbit, sheep, dog, horse, gray mouse lemur and macaque) were collected, using techniques derived from those previously used in humans and rats. The possibility of amplifying these cells and their stemness features and differentiation capability were then evaluated.ResultsBiopsies were successfully performed on olfactory mucosa without requiring the sacrifice of the donor animal, except mice. Cell populations were rapidly generated from olfactory mucosa explants. These cells displayed similar key features of their human counterparts: a fibroblastic morphology, a robust expression of nestin, an ability to form spheres and similar expression of surface markers (CD44, CD73). Moreover, most of them also exhibited high proliferation rates and clonogenicity with genus-specific properties. Finally, OE-MSCs also showed the ability to differentiate into mesodermal lineages.ConclusionsThis article describes for the first time how millions of OE-MSCs can be quickly and easily obtained from different mammalian genera through protocols that are well-suited for autologous transplantations. Moreover, their multipotency makes them relevant to evaluate therapeutic application in a wide variety of tissue injury models. This study paves the way for the development of new fundamental and clinical studies based on OE-MSCs transplantation and suggests their interest in veterinary medicine.

Initial clonogenic potential of human endothelial progenitor cells is predictive of their further properties and establishes a functional hierarchy related to immaturity

Endothelial progenitor cells (EPCs) generate in vitro Endothelial Colony Forming Cells (ECFCs) combining features of endothelial and stem/progenitor cells. Their angiogenic properties confer them a therapeutic potential for treating ischemic lesions. They may be isolated from umbilical cord blood (CB-ECFCs) or peripheral adult blood (AB-ECFCs). It is generally accepted that CB-ECFCs are more clonogenic, proliferative and angiogenic than AB-ECFCs. Nevertheless, only a few studies have focused on the functional heterogeneity of CB-ECFCs from different individuals. Moreover, AB-ECFC loss of function is yet to be precisely described. We have focused on these two issues that are critical for clinical perspectives.The detailed clonogenic profile of CB-ECFCs and AB-ECFCs was obtained and revealed a high inter individual heterogeneity and the absence of correlation with age. Most CB-ECFCs yielded initial colonies and had functional properties similar to those of AB-ECFCs. Conversely, a high clonogenicity was associated with an enhanced proliferative and angiogenic potential and stemness gene overexpression, confirming that immaturity, lost by AB-ECFCs, was a prerequisite to functionality. We thus demonstrated the importance of selecting CB-ECFCs according to specific criteria, and we propose using the initial clonogenicity as a relevant marker of their potential efficacy on vascular repair.

Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts

Human mesenchymal stromal/stem cells (MSCs) are multipotent and currently undergoing hundreds of clinical trials for disease treatments. To date, no studies have generated induced MSCs from skin fibroblasts with chemicals or growth factors. Here, we established the first chemical method to convert primary human dermal fibroblasts into multipotent, induced MSC-like cells (iMSCs). The conversion method uses a defined cocktail of small molecules and growth factors, and it can achieve efficient conversion with an average rate of 38% in 6 days. The iMSCs have much higher clonogenicity than fibroblasts, and they can be maintained and expanded in regular MSC medium for at least 8 passages and further differentiated into osteoblasts, adipocytes, and chondrocytes. Moreover, the iMSCs can suppress LPS-mediated acute lung injury as effectively as bone marrow-derived mesenchymal stem cells. This finding may greatly benefit stem cell biology, cell therapy, and regenerative medicine.

The clinical value of patient-derived glioblastoma tumorspheres in predicting treatment response.

Advances from glioma stemlike cell (GSC) research, though increasing our knowledge of glioblastoma (GBM) biology, do not influence clinical decisions yet. We explored the translational power of GSC-enriched cultures from patient-derived tumorspheres (TS) in predicting treatment response. The relationship between TS growth and clinical outcome was investigated in 52 GBMs treated with surgical resection followed by radiotherapy and temozolomide (TMZ). The effect on TS of radiation (6 to 60 Gy) and of TMZ (3.9 μM to 1 mM) was related with patients' survival. Generation of TS was an independent factor for poor overall survival (OS) and poor progression-free survival (PFS) (P < .0001 and P = .0010, respectively). Growth rate and clonogenicity of TS predicted poor OS. In general, TS were highly resistant to both radiation and TMZ. Resistance to TMZ was stronger in TS with high clonogenicity and fast growth (P < .02). Shorter PFS was associated with radiation LD50 (lethal dose required to kill 50% of TS cells) >12 Gy of matched TS (P = .0484). A direct relationship was found between sensitivity of TS to TMZ and patients' survival (P = .0167 and P = .0436 for OS and PFS, respectively). Importantly, values for TMZ half-maximal inhibitory concentration <50 μM, which are in the range of plasma levels achieved in vivo, identified cases with longer OS and PFS (P = .0020 and P = .0016, respectively). Analysis of TS holds translational relevance by predicting the response of parent tumors to radiation and, particularly, to TMZ. Dissecting the clonogenic population from proliferating progeny in TS can guide therapeutic strategies to a more effective drug selection and treatment duration.