In the SAINZlab, we study CSCs in the context of pancreatic ductal adenocarcinoma (PDAC), the 4th leading cause of cancer related deaths in developed countries. We are running a combined basic and translational research program, which synergistically combines studies on the biology of mouse and human CSCs, including their in vivo tumor microenvironment (TME), in order to enhance our understanding of the regulatory machinery of CSCs. Specifically, the avenues of research that the laboratory are pursuing include:
1) The identification and characterization of new biomarkers for the detection of CSCs from different solid tumors. We have discovered several new biomarkers present on the cell surface or within CSCs, and across several solid tumors. For example, the CSC biomarker termed autofluorescence, is the result of riboflavin accumulation in ABCG2-coated intracellular vesicles exclusively found in CSCs. We are currently using autofluorescence as a means of isolating CSCs for in-depth biological, molecular and “omics”-based characterization studies.
2) The identification of proteins that govern key CSC phenotypes, such as “stemness”, epithelial to mesenchymal transition (EMT), oxidative phosphorylation (i.e. mitochondrial respiration) and chemoresistance. For example, we have discovered that the Interferon Stimulated Gene 15 (ISG15) is not only up-regulated in CSCs, but its function as a Ubiquitin-like modifier is necessary for many CSC biological processes.
3) Comprehensively understand the cellular make-up of the CSC niche and the larger more complex tumor microenvironment (TME), specifically the role of tumor-associated macrophages (TAMs) in “activating” CSCs, with respect to the different environmental proteins they can secrete in response to cues from the tumor and how these proteins alter the function of the CSC at the level of EMT and chemoresistance.
Importantly, we would like to thank all of our past and current collaborators for facilitating many of the discoveries presented in this website. They have been and continue to be crucial for the advancement of our research.
Cancer Stem Cells (CSCs)
CSCs represent the root of the tumor, giving rise to all of the other tumor cells and driving intratumor heterogeneity. This subpopulation of cells have been shown to be exclusively tumorigenic, and highly metastatic and chemoresistant. Thus, from a clinical perspective, only elimination of the CSC will ensure tumor eradication.
By definition, CSCs are the sole source of tumor initiation, metastasis and cellular heterogeneity, giving rise to intermediate progenitors and terminally-differentiated bulk tumor cells. However, whether the CSC is a hardwired entity or a state has been a point of debate for the past 5 years. Learn more
The Cancer Stem Cell (CSC) model. CSCs share phenotypes and characteristics of normal stem cells, such as unlimited self-renewal, which assures the survival of the CSC pool and supports the hierarchical model of tumor cell heterogeneity. CSCs also possess the capacity to divide symmetrically or asymmetrically, generating more CSCs or the multiple cell lineages present within the tumor bulk, including progenitor/transient cells and more differentiated tumor cells. The degrees of pluripotency, plasticity and chemoresistance are believed to correlate with the level of cellular differentiation and degree of heterogeneity. The less differentiated and heterogenous cells possess more stem-like characteristics and are more plastic; however, progenitor/transient cells (and differentiated tumor cell to some degree) are also plastic under certain circumstances.
We now know that CSC-ness is not an intrinsic feature of a subpopulation of cells, but rather, CSC-ness is a state governed and driven by temporal and spatial characteristics, and strongly influenced by the tumor microenvironment (TME). Thus only by targeting the CSC, the non-CSC transient cells and the TME will we succeed in curing cancer.
Our laboratory is work towards this goal by trying to understand CSC plasticity and the TME signals that influence CSC-ness.
The CSC niche. CSCs are believed to reside in a niche within the tumor microenvironment (TME). The niche is a smaller anatomically distinct and more specialized TME sub-compartment, which regulates CSC fate through cell-to-cell contacts or via cues from secreted niche milieu factors. Different CSC niches can exists, and dominate at any given time during the evolution of the tumor. If the CSC is lost, there is competition for other cells to reenter the niche to replace the CSC. This process, known as neutral competition in normal stem cell niches, is where stem cell progenies respond to extrinsic signaling factors and compete to occupy the niche. The same is believed to occur in tumors. Progenitor cells, transient cells and even “committed” differentiated tumor cells can undergo phenotypic transitions and transconvert into a CSC, a process known as cellular plasticity. In addition, it is likely that other CSCs (perhaps quiescent CSCs), can reoccupy the niche; however the degree of plasticity and capacity to undergo phenotypic transitions will likely determine what cell type can reenter the niche.
CSCs can be identified and isolated via cell surface or internal markers that are over-expressed in and/or on these cells. We have identified and continue to discover new CSC markers that enable us to quantify the number of CSCs in patient biopsies as well as separate these cells away from the the bulk tumor population in order to interrogate them on a single-cell level.
Human pancreatic CSCs can be enriched for by culturing cells as spheres in anchorage-independent conditions. (A-B) CSCs within spheres can be identified using established CSC markers such as CD133 or CD44. (C) CSCs can also be identified based on the accumulation of the fluorescent vitamin B2 (green) within cytoplasmic ABCG2-coated vesicles (red). (D) Likewise, murine CSCs can be identified in pancreatic tissue using a combination of markers, such as EpCAM and CD133. (Photos kindly provided by Irene Miranda, Patrick Hermann and Susana Garcia).
CSC Autofluorescence. In 2014, we and our collaborators (Christopher Heeschen) made a novel discovery in the field of CSC biomarkers. We showed that PDAC tumors contain a subpopulation of cells with discrete intracellular autofluorescent vesicles, and these autofluorescent vesicles could be used to efficiently isolate, via flow cytometry, subsets of cells with robust CSC properties. Subsequent studies determined that the source of the autofluorescence was the consequence of riboflavin accumulation in cytoplasmic ER-derived vesicles that over express the ATP-binding cassette (ABC) transporter ABCG2. Since riboflavin is a natural substrate for ABCG2, its accumulation in these ABCG2-coated vesicles was not surprising.
Model of Pancreatic CSC autofluorescent vesicle formation. ABCG2 is translated in the ER as a six transmembrane domain nonfunctional monomer containing a NBD (nucleotide binding domain). ABCG2 is subsequently folded and glycosylated in the ER (1), and transported through the Golgi (2) to the plasma membrane where it localizes as a functional transporter (3). In general, misfolded ABCG2 undergoes ER-associated degradation (ERAD). Recently, however, it has been shown that over-expression of the E3 ubiquitin-ligase co-factor Derlin-1 can suppress ER to Golgi transport of ABCG2, resulting in ER retention. We hypothesize that in CSCs, ABCG2 is not only over expressed, but it is retained in the ER via a Derlin-1 mediated process, and as a consequence ABCG2-coated ER-derived vesicles form (4). These vesicles can then act as an intracellular sink for riboflavin (5), a natural substrate for ABCG2, resulting in the formation of the CSC autofluorescent vesicle.
CSC biomarkers. Human pancreatic cancer stem cells can be identified using cell surface markers (e.g. EpCAM, CD133, CD44, CXCR4, ABCG2, CCR7) as well as functional markers (ALDH1). We and our collaborators (Christopher Heeschen) have added to this list by identifying additional cell surface markers, such as CD47, P2X7R, FPR2 and ANTXR, as well as functional markers, such as riboflavin accumulation in ABCG2-coated vesicle (i.e. autofluorescence).
While no one marker or combination of markers can identify all of the CSC populations present within a tumor at a given time, we hope that by identifying new markers, we can fine tune our ability to identify and isolate these cells.
Our laboratory is work towards this goal by using new approaches to scan the surface of CSCs in search of new CSC markers.
The Achilles’ heel of CSCs
At the ”omic” level, CSCs are different than their non-CSC counterparts. These differences are believed to be epigenetically driven as well as a consequence of post translational modifications. We have dissected CSCs and discovered what makes them tick, and learned how to genetically and pharmacologically target these weaknesses.
Using different approaches, including high-throughput methylation analysis, mircoRNA arrays, and RNAseq, we are closer to understating what makes a CSC a CSC.
Epigenetic regulation of Cancer Stem Cells. Graphical summary of the epigenetic mechanisms regulating CSCs. DNA methylation, histone modifications and non-coding RNA molecules (lncRNAs and miRNAs) work together to modify CSC biology and plasticity.
(A) The DNMT1 methyltransferase methylates CpG sites contributing to differential methylation of genes important for stemness and differentiation. It is likely that in parallel, TET proteins generate 5hmC conferring active or passive demethylation. While several studies have shown that DNMT1 is over expressed in CSCs, the level of TET proteins in CSCs is yet to be determined. Non-coding RNA molecules, specifically miRNA and lncRNA molecules add an additional layer of complexity and regulation.
(B) miRNAs can target a number of mRNAs that play important roles as pro- or anti-CSC regulators. For example, the miR17-92 cluster targets a number of genes important for pancreatic CSC-ness, and as such the expression of this cluster is down regulated in CSCs. On the other hand, miRNAs that promote EMT features, such as the loss of cell adhesion, are upregulated in CSCs. Examples include miR-9 (target: E-cadherin), miR-194 (target N-cadherin), miR-661 (target: Nestin and Star1). mRNAs important for self-renewal, EMT and chemoresistance.
(C) lncRNA can represent a bridge between the many layers of epigenetic gene regulation, interacting with, for example, histone modifiers or serving as ceRNAs for miRNAs.
(D) Genes important for stemness, differentiation (diff.) and metabolic reprogramming are found with active and inhibiting histone tags, and histone modifying enzymes are expressed at different levels between CSCs and their non-CSCs counterparts, such asMLL1, EZH2 and KDM6A. (Center) The plasticity of differentiated cancer cells and CSCs and their ability to quickly transdifferentiate could be due to a balance between, high and/or low levels of DNA methylation, hydroxymethylation, histone acetylation or methylation, together with tumor-suppressive/oncogenic miRNAs and lncRNAs
Our laboratory continues to work towards better understanding the “omic” landscape of CSCs and non-CSCs in order to design and develop new therapeutics to specifically target CSCs
Via collaborations with national and international hospitals, we are creating one of the largest Biobanks of pancreatic patient-derived xenografts (PDXs) in Spain. PDXs are generated by implanting resected tumor pieces into immune compromised mice. The resulting “avatar” mice can serve as pre-clinical models to assess the effect of CSC inhibitors on PDAC tumor formation.
Using ”avatar” mice, we have discovered therapies that could be potentially useful and effective for treating PDAC.
The use of animals in scientific research is fundamental and necessary, but at the same time investigators must follow strict guidelines to ensure maximun welfare of animals. As such, mice are always housed according to institutional protocols and all experiments are performed in compliance with the institutional guidelines for the welfare of experimental animals approved by our Institutional Ethics Committee (IEC), Governmental bodies and in accordance with the regulations for Ethical Conduct in the Care and Use of Animals as stated in The International Guiding Principles for Biomedical Research involving Animals, developed by the Council for International Organizations of Medical Sciences (CIOMS).
We thank all of our national and international funding organizations for supporting the research conducted in the SAINZlab.