s2fieldsofstudy: Biology Medicine

text:
Testicular Niche Required for Human Spermatogonial Stem Cell Expansion

Prepubertal boys treated with high‐dose chemotherapy do not have an established means of fertility preservation because no established in vitro technique exists to expand and mature purified spermatogonial stem cells (SSCs) to functional sperm in humans. In this study, we define and characterize the unique testicular cellular niche required for SSC expansion using testicular tissues from men with normal spermatogenesis. Highly purified SSCs and testicular somatic cells were isolated by fluorescence‐activated cell sorting using SSEA‐4 and THY1 as markers of SSCs and somatic cells. Cells were cultured on various established niches to assess their role in SSC expansion in a defined somatic cellular niche. Of all the niches examined, cells in the SSEA‐4 population exclusively bound to adult testicular stromal cells, established colonies, and expanded. Further characterization of these testicular stromal cells revealed distinct mesenchymal markers and the ability to undergo differentiation along the mesenchymal lineage, supporting a testicular multipotent stromal cell origin. In vitro human SSC expansion requires a unique niche provided exclusively by testicular multipotent stromal cells with mesenchymal properties. These findings provide an important foundation for developing methods of inducing SSC growth and maturation in prepubertal testicular tissue, essential to enabling fertility preservation for these boys.

INTRODUCTION
Approximately 7,500 boys are diagnosed with cancers annually in the United States [1]. Although patients have seen significant survival improvements with treatments [2], many suffer from permanent sterilization [3,4]. For postpubertal boys and adults, fertility preservation with cryopreserved semen is highly successful [5][6][7]. Unfortunately, this option is not feasible for prepubertal boys.
Despite advances in fertility treatment that have led to the routine use of IVF/ICSI for men with ejaculated or surgically obtained sperm concentrations approaching zero, these techniques are not possible for prepubertal boys because they do not produce sperm. However, their testicles do contain SSCs [20][21][22] with the potential to expand and differentiate into mature sperm. Developing a technique to expand SSCs for autologous transplantation or to differentiate SSCs into mature sperm would be of tremendous value. Current primary challenges include the identification, isolation, and expansion of highly purified SSCs primarily because of a gap of knowledge in identifying human SSCs based on extracellular marker expression and understanding the cellular environment ("niche") necessary for SSC growth and differentiation [23][24][25][26][27].
Expansion of purified mouse SSCs using unique membrane protein markers such as THY1, GFRa1, GPR125, and CD49f has been reported [23,[28][29][30]. Allogeneic transplantation of in vitro expanded mouse SSCs into germ cell-depleted testes resulted in restoration of fertility [31]. Recent studies propose that human SSCs may be identified based on expression of THY1, CD49f, EpCAM, and SSEA-4 [32][33][34][35]. Of these markers, only SSEA-4 is highly expressed in embryonic stem cells and in both human fetal and prepubertal SSCs, suggesting that it may be a good marker of adult human SSCs [20,36]. Although transplantation of human testicular cells into germ cell-depleted mouse testes resulted in either limited colonization of human cells or cells expressing germ cell markers, spermatogenesis was not detected, presumably because of interspecies differences [33-35, 37, 38]. Interestingly, mouse and human SSCs have been reported to have the capability of converting into testis-derived pluripotent stem cells during in vitro culture [33,34,[39][40][41][42][43]. However, in contrast to studies in mice, recent studies suggest that the human testis-derived pluripotent stem cells derived from in vitro culture of putative human SSCs are actually cells of mesenchymal rather than germ cell origin. Filling this gap in our understanding of human SSC biology is critical [24][25][26][27].
Because of the limited availability of human tissues, the lack of in vitro or in vivo xenograft models capable of supporting human spermatogenesis, and the significant ethical and logistical challenges associated with human germ cell research, current data on the identification, isolation, and expansion of human SSCs are mixed and highly controversial. To shed light on this controversy and lay the groundwork for a new therapy for young male patients facing sterilizing treatments, a detailed characterization of SSCs and the required somatic niche capable of supporting SSC expansion is required [44].
In this study, we characterize the cellular niche required for the expansion of human SSCs, provide strong evidence supporting specific extracellular markers that define a testicular cell population highly enriched for human SSCs, and characterize a novel testicular multipotent stromal cell (TMSC) population essential for SSC expansion. The results of this study are very important to understanding how human SSCs can be grown and expanded successfully in vitro for future therapeutic applications.

Subjects
Thirteen adult subjects with normal spermatogenesis were enrolled in the institutional review board-approved University of California San Francisco LIFE and Human SSC studies.
RNA-seq data were generated from fluorescence-activated cell sorting (FACS)-sorted human testicular THY1+ cells and SSEA-4+ cells obtained from three healthy individuals. Briefly, THY1+ and SSEA-4+ cells were sorted directly into RNA buffer. Purified RNA was analyzed for quality using chip-based capillary electrophoresis (Bioanalyzer; Agilent Technologies, Palo Alto, CA, http://www.agilent.com), and quantity and purity were determined with a NanoDrop spectrometer. RNA-seq libraries were prepared with an ovation RNA-seq system v2 kit (NuGEN, San Carlos, CA, http://www.nugen.com/nugen). In this method, the mRNA is reverse transcribed to synthesize the first-strand cDNA using a combination of random hexamers and a poly-T chimeric primer. The RNA template is then partially degraded by heating, and the second strand cDNA is synthesized using DNA polymerase. The double-stranded DNA is then amplified using single primer isothermal amplification (SPIA) [45,46]. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 59-end of the double-stranded DNA, after which the SPIA primer binds to the cDNA and the polymerase starts replication at the 39-end of the primer by displacement of the existing forward strand. Random hexamers are then used to amplify the second-strand cDNA linearly. Finally, libraries from the SPIA-amplified cDNA were made using the Ultralow DR library kit (NuGEN). The RNA-seq libraries were analyzed by Bioanalyzer and quantified by qPCR (KAPA, Wilmington, MA, http:// www.kapabiosystems.com). High-throughput sequencing was done using a HiSEquation 2500 (Illumina Inc., San Diego, CA, http://www.illumina.com).

SSC Culture
Subpopulations of testicular cells were FACS-sorted and individually cultured in supplemented StemPro-34 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) [31,34] with the following modifications: 1% penicillin-streptomycin, 1% insulin-transferrinselenium (ITS) solution (Mediatech Inc., Manassas, VA, http:// www.cellgro.com), recombinant human epidermal growth factor (20 ng/ml) (R&D Systems Inc., Minneapolis, MN, http://www. rndsystems.com), recombinant human glial cell line-derived neurotrophic factor (GDNF) (10 ng/ml) (R&D Systems), recombinant human leukemia inhibitory factor (LIF) (10 ng/ml) (Chemicon, Temecula, CA, http://www.chemicon.com) and 1% knockout serum replacement (KOSR). The medium was changed every 72 hours. Cells were cultured at 37°C in a humidified incubator with 5% CO 2 and 20% O 2 without stromal cells on either Matrigel (BD Biosciences, San Diego, CA, http://www.bdbiosciences.com)coated dishes or 0.2% gelatin-coated dishes at a density of 50,000 cells per cm 2 and on either mouse embryonic fibroblasts (MEFs), human fetal placental fibroblasts, human fetal testicular fibroblasts, or human adult testicular THY1+ cells at a density of 25,000 cells per cm 2 . Human fetal placental and testicular fibroblasts at 22 weeks of gestation were generated from discarded fetal tissues donated for research.  [20,23,33,35]. However, although recent studies demonstrate that both THY1 and SSEA-4 are expressed in fetal gonocytes, only SSEA-4 continued to be expressed in prepubertal SSCs [20]. Thus, confocal microscopy was performed to detect the presence and precise location of cells expressing THY1, SSEA-4, and VASA. Two distinct populations of germ cells were observed by relative expression of VASA (Fig. 1A). VASA dim cells, located at the basement membrane, correlate anatomically with the known location of SSCs. In contrast, VASA bright cells, located predominantly away from the basement membrane toward the lumen, are consistent with differentiating spermatogonia, spermatocytes, and spermatids. SSEA-4 expression was primarily detected in VASA dim cells on the basement membrane, suggesting that it is a specific marker for SSCs (Fig. 1A). Additionally, evidence of meiosis, assessed by SYCP3 expression, was exclusively detected in VASA bright cells, suggesting that the SSEA-4/VASA dim population contains adult human SSCs (Fig. 1B). In contrast, THY1 expression was detected primarily on fibroblasts and myoid cells of the lamina propria with limited expression on peritubular cells (Fig. 1C). Additionally, the cell population located between the VASA dim and bright cells exclusively expressed both THY1 and WT1, consistent with Sertoli cells (Fig. 1C). All THY1+ cells expressed high levels of vimentin and did not express VASA, suggesting that they are likely of somatic and mesenchymal origin (Fig. 1D).

Characterization of the Niche Required for SSC Expansion Testicular THY1+ Cells Are Critical for Successful SSC Expansion
Unsorted, sorted THY1+, and sorted SSEA-4+ cells were subjected to in vitro expansion and monitored with time-lapse photography (supplemental online Videos 1-4). Unsorted testicular cells cultured on either uncoated or coated plates revealed two populations. The first adhered to the plates and exhibited fibroblast-like morphology within 48 hours. The second population of small round cells bound to these fibroblast-like adherent cells shortly after 48 hours, divided, and formed colonies after 2 weeks of culture ( Fig. 4A). However, colonies began to disappear after 3 weeks of culture because the adherent cells became confluent (supplemental online Video 1). Although ∼98% of these in vitro expanded unsorted testicular cells expressed THY1, evaluated by FACS, after 3 weeks of culture, neither SSEA-4 nor VASA expression was detected by FACS, microscopy, or qPCR. Cell passage after 2 weeks of culture did not rescue expansion of SSC colonies because the adherent cells quickly grew to confluence, suggesting a preferential selection of THY1+ cells in this culture system.
When plated on culture dishes uncoated or coated with either Matrigel or gelatin, THY1+ cells adhered to all plates within 24 hours, exhibited fibroblast morphology shortly after, and continued to expand without signs of quiescence (.20 passages) ( Fig.  4A; supplemental online Video 2). Although DAZL and VASA were never detected by qPCR or confocal microscopy, this population continued to express high levels of THY1 and vimentin, assessed by immunofluorescent analyses. In contrast, SSEA-4+ (Fig. 4A) and THY12/SSEA-42 cells did not adhere or form colonies when cultured on uncoated or coated plates, failed to expand, and died within 2 weeks of culture. Furthermore, immunofluorescent analyses did not detect any evidence of THY1 and vimentin expression in these two populations.
To overcome the rapid expansion of THY1+ cells in this system, sorted THY1+ cells were expanded and subjected to g-irradiation to render them mitotically inactive. Sorted SSEA-4+ cells were then cocultured on the irradiated adherent THY1+ cells. SSEA-4+ cells bound to these adherent cells within 24 hours, formed SSC colonies (∼50 cells per colony) within 2 weeks, and continued to expand ( cells uniquely provide the essential niche required for SSC expansion. Using this novel system, SSC colonies were successfully identified, isolated, passaged, and expanded in vitro.

Testicular THY1+ Cells Demonstrated Mesenchymal Properties
In addition to their lack of germ cell properties, sorted THY1+ cells immediately adhered to plastic and exhibited fibroblastic morphology, suggesting a mesenchymal origin. To investigate whether the THY1+ population exhibited mesenchymal characteristics, THY1+ cells were analyzed for coexpression of CD73 and CD105. 92% of THY1+ cells coexpressed both CD73 and CD105 (Fig. 5A). Of note, CD45+ cells were excluded from analyses in the SYTOX Blue gating using anti-CD45 conjugated with Pacific Blue. When THY1+ cells were expanded in vitro, they continued to coexpress both CD73 and CD105 (Fig. 5B).
Comprehensive Molecular Characterization of the Testicular THY1+ and SSEA-4+ Cells by Next Generation Sequencing Testicular SSEA-4+ Cells Expressed Genes Previously Identified as Enriched in Human and Mouse SSCs FACS-sorted THY1+ and SSEA-4+ cells were collected from three patients and subjected to mRNA sequencing. On average, there were 13,568,327 and 8,822,058 total reads from the THY1+ and SSEA-4+ populations, respectively. The principal component analysis is shown in Figure 6A. As demonstrated, the THY1+ and SSEA-4+ populations are significantly distinct from each other. Although the SSEA-4+ population clusters tightly, the THY1+ population shows more variability, reflecting this population's innate heterogeneity. When log 2 6 twofold change with a p value of ,0.05 was used as the cutoff to define significant differential gene expression, there were 1,359 known upregulated and 1,911 downregulated genes in the SSEA-4+ population in comparison with the THY1+ population. Specifically, 29 genes were upregulated, and 232 were downregulated .100-fold (supplemental online Tables 1, 2). Genes previously reported to be enriched in human and mouse SSCs were examined. Table 1 demonstrates the enriched expression of known SSC genes in testicular SSEA-4+ cells. When further evaluated by FACS, EPCAM, GPR125, and ITGA6/CD49f were found to express in both THY1+ and SSEA-4+ populations (Table 1). However, qPCR confirmed the higher expression of GPR125 in the SSEA-4+ population (Fig. 3B). Although KIT was differentially expressed in the SSEA-4+ population, C-Kit was not detected by confocal microscopy or FACS. Known intracellular markers of SSCs were highly enriched in the SSEA-4+ cells. Although ZBTB16 was not found to be enriched with mRNA-seq, qPCR data demonstrated that it was significantly enriched (1.9fold) in the SSEA-4+ population (Fig. 3B). Additionally, known genes (RET, GFRa1, and ETV5) in the GDNF-mediated SSC selfrenewal pathway in rodent were also highly enriched in SSEA-4+ cells (Table 1; Fig. 3B) [47]. Furthermore, known pluripotency markers such as TERT and LIN28B were highly expressed in the SSEA-4+ cells, further suggesting that this population contains human SSCs. As expected and confirmed with qPCR, NANOG, SOX2, and POU5F1 were not expressed in any significant amount in either populations. In contrast, known somatic genes were highly expressed in the THY1+ population ( Table 1). The diverse families of somatic genes expressed in the THY1+ cells confirmed that this is a heterogeneous population. Overall, the testicular SSEA-4+ and THY1+ mRNA transcriptome profiles confirmed that the SSEA-4+ population contains primitive spermatogonia in contrast to the profile of the THY1+ population that favors a somatic origin.

Testicular SSEA-4+ Cells Expressed Genes Previously Identified as Enriched in Human Prepubertal SSCs and Mouse Gonocytes
Because the results thus far support the hypothesis that testicular SSEA-4+ population contains adult human SSCs, it is important to compare this population with known pure populations of human or mouse SSCs. Hence, the transcriptome of the testicular SSEA-4+ cells reported here was compared with previously published human prepubertal SSC transcriptome because current information on adult human SSCs is limited and controversial. When log 2 6 twofold-change with a p value of ,0.05 was used to define significant differential gene expression, there were 798 genes that were upregulated and 251 that were downregulated in the human prepubertal SSCs in comparison with their respected somatic population with the top 50 up-and downregulated genes shown in supplemental online Table 3. In contrast, 112 genes were upregulated and 13 were downregulated in the mouse gonocytes in comparison with their somatic populations (supplemental online Table 4). There were 24 common upregulated genes found between the three groups ( Fig. 6B; supplemental online Table 5). Mouse gonocytes shared 35% (44 of 125) of their differentially expressed genes with human prepubertal SSCs (Fig. 6B). In contrast, human prepubertal SSCs and mouse gonocytes shared 42% (443 of 1,049) and 43% (54 of 125) of their differentially expressed genes, respectively, with SSEA-4+ cells (Fig. 6B). Alternatively, when only the top 50 upregulated genes from the human prepubertal SSCs and mouse gonocytes were evaluated, 68% (34 of 50) and 54% (27 of 50) of the differentially expressed genes, respectively, were in common with differentially upregulated genes seen in human testicular SSEA-4+ cells. Thus, the similarity between the transcriptome profiles of human testicular SSEA-4+ cells, human prepubertal SSCs, and mouse gonocytes further demonstrates that human adult SSCs are within the testicular SSEA-4+ population.

DISCUSSION
Developing the ability to isolate SSCs and understanding the testicular niche optimal for SSC growth and development are required to develop effective therapeutic options for pediatric cancer patients facing sterilizing treatments. Using confocal microscopy, FACS-sorted subpopulations of testicular cells, timelapse photography, comprehensive mRNA sequencing, and a novel in vitro culture system, this study identifies a subpopulation of adult human testicular cells highly enriched for SSCs and the support cells critical to their growth based on distinct extracellular markers SSEA-4 and THY1, respectively (supplemental online Table 6). These insights provide valuable information for the development of future treatments to preserve and restore fertility.
Both testicular THY1+ and SSEA-4+ cells have been reported to contain SSCs based on mouse and human studies [23,33,35]. Specifically, transplantation of both enriched human testicular THY1+ [33] and SSEA-4+ [35] cells into mouse testes resulted in germ cell colony formation. These findings are controversial, however, because in vitro culture of human unsorted testicular cells and enriched THY1+ cells with the current system did not select for long-term SSC expansion [24][25][26][27]. Rather, recent human studies suggest that the in vitro systems selected for cells of mesenchymal origin [24][25][26][27]. Our data demonstrated that SSC colonies disappeared as THY1+ cells expanded in culture over time.
Using confocal microscopy, we demonstrated that THY1+ cells were predominantly located in lamina propria with some expression on Sertoli cells. Sorted THY1+ cells did not express germ cell markers as evaluated by microscopy (VASA) or qPCR (DAZL and VASA), suggesting a somatic origin. In contrast, previous studies demonstrated that human THY1+ populations contain SSCs after mouse xenotransplantation [33]. It is possible that this observation was the result of germ cell contamination, given that an enriched rather than sorted cell population was analyzed. When sorted testicular THY1+ cells were used to establish mRNA profile by RNA sequencing, the THY1+ transcriptome was consistent with Additionally, the transcriptome profiles of the THY1+ and SSEA-4+ populations were quite distinct, as shown in the principal component analysis (PCA) (Fig. 6A). Further functional evidence that supports sorted testicular THY1+ cells as the population containing TMSCs includes rapid binding to plastic; expression of consensus mesenchymal markers (CD73 and 105); the ability to differentiate into adipocytes, chondrocytes, and osteocytes; and the absence of CD45 expression. Given the anatomic location of the THY1+ cells within the seminiferous tubules, the lack of germ cell markers, high expression of vimentin, and the ability to differentiate into all mesenchymal lineages, the TMSC (THY1+) population, demonstrated here, is of somatic origin and not germ cells.
SSEA-4 is a marker of undifferentiated pluripotent human embryonic stem cells, cleavage to blastocyst stage embryo, human fetal SSCs, and prepubertal SSCs [20,36]. Anatomically, SSEA-4+ cells are located predominantly at the basement membrane, suggesting that they are SSCs. This is in contrast to previous studies that demonstrated limited coexpression of THY1 and SSEA-4 on subpopulations of human testicular cells using conventional microscopy [35]. However, these earlier studies did not use multicolor FACS or confocal microscopy analyses. In our studies, multicolor FACS, confocal microscopy, and transcriptomes on sorted cells confirmed that THY1+ and SSEA-4+ cells were two distinct populations with different physical, cellular, and molecular profiles. When the tubules were stained for VASA, VASA bright (SSEA-42) cells contained haploid cells and located toward the lumen, whereas the VASA dim (SSEA-4+) cells were found at the basement membrane. This finding is consistent with previous human studies demonstrating that a high level of VASA expression was associated with maturing germ cells [48,49]. In contrast to the absence of markers associated with meiosis (DMC1 and SYCP3) or differentiating spermatids (PRM2 and ACR), SSEA-4+ cells expressed high levels of putative SSC markers (ZBTB16, GFRa1, SALL4, MAGEA4, and GPR125), and pluripotency genes (TERT and LIN28B), consistent with primitive spermatogonia containing SSCs. Lower levels of ZBTB16, GFRa1, and GPR125 expression were also detected in THY1+ cells in comparison with SSEA-4+ cells in these studies. This is similar to previous human and mouse studies in which ZBTB16, GFRa1, and GPR125 are markers of SSCs, but low-level expression was also detected in testicular somatic cells [22]. Of note, the genes (GFRa1, RET, and ETV5) involved in the GDNF-mediated SSC self-renewal were also highly expressed in the SSEA-4+ cells [47]. These results support previous studies demonstrating germ cell colonization in mouse xenograft model following transplantation of primary SSEA-4+ sorted cells [35]. Interestingly, recent studies demonstrate that human bone marrow-derived mesenchymal stem cells (BmMSCs) also express SSEA-4 but lack CD45 expression [50]. Although CD45+ cells were excluded from any FACS analyses, we cannot eliminate the possibility of BmMSC contamination. Perhaps the small number of BmMSCs contributed to a very low level of VIM expression detected in our SSEA-4+ population. However, sorted SSEA-4+ cells failed to survive in vitro in the absence of other cell types, suggesting that this possible contamination is very low at best. The significant similarity in differentially expressed genes in human testicular SSEA-4+/THY1+ in comparison with human prepubertal SSCs/somatic cells and mouse gonocytes/somatic cells further solidify that human adult SSCs reside with the SSEA-4+ population [22]. Interestingly, of the 24 common upregulated genes between the 3 groups, 16 genes (ASF1B, ASPM, BUB1, CASC5, CENPA, CENPF, CENPO, EXO1, HELLS, KIF11, KNTC1, MCM8, RAD51AP1, RAD54B, STAG3, and TOP2A) are involved cell cycle, DNA replication, meiosis, and DNA repair regulations, as demonstrated in the interactive pathway analysis (supplemental online Fig. 1). Of the 24 common genes, 3 (DAZL, PIWIL4, and BNC1) are intricately involved with germ cell maintenance and  [51][52][53]. When the top 50 differentially upregulated genes from human prepubertal SSCs were compared with human testicular SSEA-4+ cells, 19 of the 34 commonly upregulated genes (STK31, MAGEA4, TPTE, VASA, TKTL1, MAEL, ELAVL2,  LIN28B, TEX15, SNAP91, CENPE, SLC25A31, DPPA2, FGFR3,  DPPA4, DAZL, CASC5, TOP2A, and CENPF) are also involved in pluripotency, cell cycle, meiosis, DNA repair, and germ cell regulations [54][55][56][57][58][59][60][61][62][63]. Of the 16 nonoverlapping genes, SOHLH2 and POLB were significantly upregulated in human SSEA-4+ population but at ,log 2 twofold change. Of the 14 remaining nonoverlapping genes, only 1 (CD109) has been well characterized [64]. CD109 is well characterized in the hematopoietic system, and its expression is likely due to contamination with the prepubertal SSC isolation because the SSCs were manually isolated [22]. In contrast, when the top 50 upregulated genes in the mouse gonocytes were compared with human testicular SSEA-4+ cells, 20 of the 27 commonly upregulated genes (EPCAM, BNC1, CENPF, CDCA7L, DAZL, TOP2A, STAG3, MCM8, BUB1, RAD54B, KNTC1, EXO1, CASC5, PIWIL4, CDCA5, SALL4, RAD51, ERCC6L, TPX2, and CDCA2) are involved in the pluripotency, cell cycle, meiosis, DNA repair, and germ cell regulations [65,66]. Thus, these findings highlight the evolutionary conserved genes in SSCs that are essential for reproduction.
When sorted THY1+, SSEA-4+, and THY12/SSEA-42 cells were cultured separately on either uncoated or coated plates, only THY1+ cells adhered and grew in a monolayer fashion regardless of surface substrate. In contrast to the lack of germ cell expression, THY1+ cells continued to express THY1+/CD73+/CD105+ and vimentin after more than 20 passages, further confirming a mesenchymal origin. When unsorted testicular cells were cultured, germ cells quickly bound to the adherent cells, formed colonies within 2 weeks of culture, and continued to express SSEA-4 and VASA. However, the adherent cells quickly outgrew and subsequently inhibited the SSC colonies growth by 4 weeks of culture. To delineate the relationship between SSC growth and its required niche, sorted SSEA-4+ cells were cultured in the presence of irradiated MEFs, human placental fibroblasts, human fetal testicular fibroblasts, and sorted adult testicular THY1+ cells. SSEA-4+ cells bound exclusively to testicular THY1+ cells within 48 hours and established colony formations. Under these conditions, SSC colonies continued to expand in size and underwent many subsequent passages without loss of SSEA-4 and VASA expression. This observation is consistent with recent studies that have demonstrated the importance of testicular stromal cells as a source of essential growth factors required by SSCs [67]. Previous studies demonstrated the presence of both SSCs and testis-derived pluripotent stem cells when unsorted testicular cells were cultured in vivo [34]. Thus far, we have not observed this phenomenon with either unsorted testicular cells or sorted testicular SSEA-4+ cells. All observed colonies in our studies expressed SSEA-4 and VASA. Although the percentage of SSC colonies formed per sorted SSEA-4+ cells (25,000 cells per experiment) appeared to be low (0.02%-0.1%) using our system, the cell number required for engraftment of SSEA-4+ colonies using a mouse model was more than 300,000 cells per transplant [35]. This suggests that the SSEA-4+ population is enriched for SSCs and that only a small portion of the population can form colonies and repopulate in vivo. The number of germ cells needed for engraftment in autologous nonhuman Smith, Yango, Altman et al.
primate transplant model was ∼100 3 10 6 cells [9]. Thus, the low initial concentration of SSCs in testicular tissue highlights the need for in vitro expansion of SSCs prior to developing clinically viable SSC transplantation techniques in the future. Although MEFs were found to support in vitro mouse SSC expansion [31], this was not the case with human SSCs, suggesting basic differences between human and mouse SSCs. These data provide strong evidence that SSEA-4 is a specific marker expressed in primitive SSCs, whereas the somatic THY1+ cells are TMSCs that play an instrumental role in providing the appropriate niche required for SSC expansion.
A weakness of the study includes the lack of SSEA-4+ cell xenotransplantation, before and after in vitro expansion [11]. Although allotransplantation of mouse SSCs into germ cell-depleted testes is an ideal in vivo assay to evaluate for the ability of mouse SSCs to rescue spermatogenesis, it is suboptimal for human SSCs. Presumably because of interspecies differences, xenograft of human SSCs into mouse testes resulted in only colonization of human cells without differentiation. Furthermore, recent studies demonstrate that allogeneic mesenchymal stem cell (MSC) xenotransplantation also resulted in formation of germ cell colonies, highlighting this model's lack of specificity, especially in the absence of in vivo differentiation [68]. Thus, there is no current ideal in vivo assay to evaluate human SSC activity.
These data have many significant strengths. Using a combination of multicolor FACS, confocal microscopy analyses, molecular profiling, and cell culture with sorted subpopulations of testicular cells monitored with time-lapse photography, we were able to definitively identify and differentiate the stromal and SSC compartments within the human testicular niche. Of critical importance was the finding that specific interactions between adult testicular THY1+ cells and SSCs are essential for in vitro human SSC expansion. Furthermore, these data likely explain the controversy regarding MSC expansion with the traditional in vitro culture system using testicular cells.

CONCLUSION
Identifying and growing SSC have important implications for patients. To date, prepubertal males facing sterilizing chemotherapy do not have a proven means of protecting their fertility. In a small number of centers around the world, testicular biopsies are performed prior to chemotherapy in the hopes that new fertility restoring treatments will become available. Development of methods to expand purified SSCs exponentially, free of malignant cell contamination, for future autologous transplant or to differentiate prepubertal SSC to mature sperm has enormous therapeutic potential. We have characterized the essential steps of isolating and expanding highly purified human SSCs using a defined somatic niche provided by the TMSCs. This is a critical step forward in developing strategies of prepubertal SSC expansion and autologous SSC therapy for fertility preservation treatments.

ACKNOWLEDGMENTS
We acknowledge Dr. Grace Wei for her assistance with time-lapse photography. Funding for the study design and conduct of the study and the collection, management, analysis, and interpretation of the data was provided in part by an American Society of Reproductive Medicine New Investigator Award and through institutional and departmental funding.

AUTHOR CONTRIBUTIONS
J.F.S. and N.D.T.: conception and design, administrative and financial support, provision of study materials or patients, data analysis and interpretation, manuscript writing, final approval of manuscript; P.Y. and E.A.: data analysis and interpretation, collection and assembly of data, manuscript writing, final approval of manuscript; S.C.: provision of study materials or patients, data analysis and interpretation, manuscript writing, final approval of manuscript; A.P., A.M.Z., M.R., and P.C.K.: data analysis and interpretation, manuscript writing, final approval of manuscript.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
M.R. has an uncompensated consultancy with Ovascience.


text:
Heterogeneous EGFR Gene Copy Number Increase Is Common in Colorectal Cancer and Defines Response to Anti-EGFR Therapy

Anti-EGFR therapy is commonly used to treat colorectal cancer (CRC), although only a subset of patients benefit from the treatment. While KRAS mutation predicts non-responsiveness, positive predictive markers are not in clinical practice. We previously showed that immunohistochemistry (IHC)-guided EGFR gene copy number (GCN) analysis may identify CRC patients benefiting from anti-EGFR treatment. Here we tested the predictive value of such analysis in chemorefractory metastatic CRC, elucidated EGFR GCN heterogeneity within the tumors, and evaluated the association between EGFR GCN, KRAS status, and anti-EGFR antibody response in CRC cell lines. The chemorefractory patient cohort consisted of 54 KRAS wild-type (WT) metastatic CRC patients. EGFR GCN status was analyzed by silver in situ hybridization using a cut-off value of 4.0 EGFR gene copies/cell. KRAS-WT and KRAS mutant CRC cell lines with different EGFR GCN were used in in vitro studies. The chemorefractory CRC tumors with EGFR GCN increase (≥4.0) responded better to anti-EGFR therapy than EGFR GCN (<4.0) tumors (clinical benefit, P = 0.0004; PFS, HR = 0.23, 95% CI 0.12–0.46). EGFR GCN counted using EGFR IHC guidance was significantly higher than the value from randomly selected areas verifying intratumoral EGFR GCN heterogeneity. In CRC cell lines, EGFR GCN correlated with EGFR expression. Best anti-EGFR response was seen with KRAS-WT, EGFR GCN = 4 cells and poorest response with KRAS-WT, EGFR GCN = 2 cells. Anti-EGFR response was associated with AKT and ERK1/2 phosphorylation, which was effectively inhibited only in cells with KRAS-WT and increased EGFR GCN. In conclusion, IHC-guided EGFR GCN is a promising predictor of anti-EGFR treatment efficacy in chemorefractory CRC.

Introduction
Epidermal growth factor receptor (EGFR) signaling is commonly activated in colorectal cancer (CRC). EGFR-targeting monoclonal antibodies (mAb) have become a standard treatment option, particularly in the chemorefractory phase of metastatic disease [1]. KRAS, a signaling molecule downstream of EGFR, is mutated in approximately 40% of CRCs [1] and these activating mutations convey anti-EGFR treatment resistance [2]. In KRAS wild-type (WT) tumors, objective response is achieved in every third patient indicating that other factors contribute to drug efficacy [3]. Thus, there is urgent need for novel predictive markers.
EGFR gene copy number (GCN) increase has been linked to anti-EGFR treatment response. Most studies have shown an association between GCN level and clinical benefit, progression free survival (PFS), and in some cases, with overall survival (OS) [4][5][6]. However, EGFR GCN is not currently utilized in the clinical context because of technical obstacles and considerable variation between the scoring systems [7]. We recently reported a novel algorithm, which may improve the predictive value of EGFR GCN. We first showed that the EGFR GCN as analyzed by silver in situ hybridization (SISH) positively correlated with immunohistochemistry (IHC), when the evaluation was performed from tumor areas of highest staining intensity [8]. We further demonstrated that an increased EGFR GCN, using cut-off value ($4.0), correlated positively with response to anti-EGFR therapy in all three parameters analyzed: clinical benefit, PFS, and OS. EGFR GCN was independent of KRAS status, and when the two analyses were combined, they predicted treatment response better than either test alone. The mean EGFR GCN, as analyzed by this method was 5.5, and copy number increase $4.0 was seen in 64% of the tumors. The GCN increase was typically associated with Chromosome 7 polysomy, whereas high level amplification was rarely seen.
A reason for the variation in published EGFR GCN results may be tumor heterogeneity, which has not been addressed in earlier studies. There is some evidence that EGFR may be heterogeneously expressed within individual colorectal tumors, both at gene and protein level [5]. Heterogeneity may complicate the analysis of EGFR protein expression and copy number alterations and lead to poor test reproducibility [7,9]. This may be especially relevant in FISH-based analysis, where the GCN counting cannot be correlated with histology [7]. If EGFR heterogeneity plays a biological role, then an algorithm, in which protein expression (IHC) guides EGFR GCN evaluation, could improve the predictive value.
The aim of this study was to test the novel EGFR GCN method in an independent patient cohort and to assess the impact of EGFR GCN status with outcome in a combined chemorefractory patient cohort. In both patient cohorts an EGFR increase ($4.0) was shown to associate with an improved clinical outcome, including clinical benefit rate, PFS, and OS. Secondary aims were to elucidate EGFR GCN heterogeneity within the tumors and to test, whether CRC cell lines with various EGFR GCN, respond differently to EGFR mAbs. According to our results, EGFR GCN is heterogeneous in CRC and the values obtained with IHC guidance from selected tumor areas are higher than the ones obtained by random selection. Importantly only the EGFR GCN counted with EGFR IHC guidance was able to predict response to anti-EGFR treatment. Our in vitro studies support our clinical findings, since the best response to anti-EGFR treatment was seen in KRAS WT, EGFR GCN = 4.0 cells and poorest response in the KRAS WT, EGFR GCN = 2 cells.

Patients
The original Turku University Hospital discovery cohort has been reported [8]. The validation cohort consisted of 31 KRAS-WT patients treated with EGFR mAbs in second to sixth line at the Helsinki University Hospital for metastatic CRC between June 2008 and July 2010. The selection criteria for this study were: (I) tissue was available from the primary tumor at diagnosis prior to start of any treatment, (II) the tumor was KRAS-WT, (III) the patients received second-to sixth-line treatment with cetuximab or panitumumab with or without chemotherapy, and (IV) the patients had no other malignancy in their history. The histology of each validation cohort case was re-evaluated by an expert in GIpathology (AR).
The combined cohort of chemorefractory patients included 54 patients, 25 from the original Turku cohort and 29 from the Helsinki validation set. The chemorefractory patient cohort included patients treated with anti-EGFR mAbs in third line or more, either as single therapy or in combination with irinotecan +/2 a fluoropyrimidine. Key characteristics of all three cohorts are described in Table 1.
The response to anti-EGFR treatment was evaluated by computed tomography (CT) or magnetic resonance imaging (MRI) according to the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) [10]. Clinical benefit was considered partial response (PR) or at least 3 months of stable disease (SD). Progression free survival (PFS) was calculated from the onset of anti-EGFR treatment until disease progression. Overall survival (OS) was calculated from the onset of anti-EGFR therapy until death of any cause.

Ethics Statement
The study was conducted in accordance with the Declaration of Helsinki. The clinical data were retrieved and histological samples collected and analyzed with the endorsement of the National Authority for Medico-Legal Affairs as well as the Institutional Review Board of the Hospital District of Southwest Finland and Ethical Review Board at Helsinki University Hospital. Written or oral informed consent was not obtained due to the fact that a large portion of the patients included in this retrospective study had died of their disease. The need for informed consent from participants was waived by the National Authority for Medico-Legal Affairs.

IHC and SISH Procedures
KRAS mutation analysis was performed with the DxS KRAS mutation kit (DxS Ltd, Manchester, UK). Detailed methods for EGFR IHC and EGFR GCN have been described [8]. In brief, three mm sections were first stained with EGFR (clone 5B7) mAb (Ventana Medical Systems/Roche Diagnostics, Tucson AZ, USA). Stainings were performed with BenchMark XT (Ventana/Roche) using ultraVIEW Universal DAB Detection Kit (Ventana/Roche). EGFR gene was detected from subsequent five mm sections with EGFR DNA Probe (Ventana/Roche) and ultraVIEW SISH Detection Kit (Ventana/Roche). In each tumor, EGFR GCN of forty tumor cells was analyzed using a 40x objective by two observers (ML, JS) from areas of highest IHC reactivity. The investigators were blinded of the clinical information. To evaluate the average EGFR GCN within each tumor, five tumor areas were arbitrarily chosen. From each of these areas, EGFR GCN of 20 randomly selected cancer cells was counted by two observers (TA, JS). The results were reported as mean and range within each area.

Cell Lines, Western Blotting and Cytotoxicity Assays
The C2BBe1, SK-CO-1 and NCI-H747 cell lines were purchased from ATCC (Manassas, VA, USA) and the CW-2 cell line from RIKEN bioresource center (Tsukuba, Japan). The NCI-H747 and CW-2 cells were cultured in RPMI-1640, the SK-CO-1 cells in EMEM and C2BBe1 cells in DMEM supplemented with 0.01 mg/ml human transferrin (Sigma, Saint Louis, MO, USA). All media were supplemented with 10% FBS, 2 mM glutamine and 1% penicillin/streptomycin.
For the signaling pathway analysis cells were grown on 6-wellplates and allowed to attach for 12 hours in normal medium. Then they were changed into medium with 1% FBS and given 0-200 mg/ml cetuximab (Erbitux, Merck Serono) for 24 hours. 25 mg/ml EGF was given to the cells for five minutes before lysis.
For cell viability assays, 5000 cells/well were plated on 96-well plates. After overnight incubation in the presence of 2% FBS, the cells were exposed to 0-200 mg/ml cetuximab (Erbitux, Merck Serono) or panitumumab (Vectibix, Amgen) for 72 hours in medium supplemented with 1% FBS. Each treatment was done in triplicate and the experiment was repeated four times. Cell viability was assessed with the CellTiter 96 Aqueous One Solution Cell Proliferation assay (Promega, Madison, WI, USA). The cell viability is given as percentage of the control cells.

Statistical Analysis
Statistical analyses were performed with the SAS 9.2 and Enterprise Guide 4.2 programs (SAS Institute Inc., Cary, NC).
Frequency table data were analyzed with the x2-test or Fisher's exact test. The difference in EGFR GCN values obtained by different evaluation methods (normally distributed variables) was calculated with the Students t-test. Kaplan-Meier and log-rank tests as well as Cox proportional hazards regression model were used for univariate survival analysis. All statistical tests were twosided. P-values,0.05 were considered to be statistically significant. The statistical significance for the cell viability assays was calculated with Microsoft Excel 2011 and StatPlus:mac LE (Version 2009, AnalystSoft Inc.). The significance between the differences in the responses in the cell lines was determined with two-way ANOVA followed by multiple t-tests.

EGFR GCN Test Validation with an Independent Patient Cohort
To validate our earlier results on the association between EGFR GCN and anti-EGFR treatment response, we studied an independent patient cohort treated at the Helsinki University Hospital. The methods for EGFR GCN detection and the cut-off value for GCN increase were identical to the discovery study [8].

Correlation between EGFR GCN and Treatment Response in Chemorefractory Patients
We combined the chemorefractory KRAS WT patients from both cohorts (n = 54) for further statistical analyses. Eighty per cent (28 out of 35) of the patients with a high EGFR GCN ($4.0) achieved clinical benefit. In contrast, the clinical benefit rate was only 32% (6 out of 19) for the patients with a low EGFR GCN (, 4.0) (Chi-Square test, P = 0.0004).
We analyzed separately the 31 chemorefractory patients treated with cetuximab (57%) and 21 with panitumumab (39%). Two patients were treated with both anti-EGFR antibodies sequentially and therefore excluded from the analysis. The clinical benefit rate in the patients treated with cetuximab +/2 cytotoxic therapy with a high EGFR GCN in their primary tumors was 86% (18/21) as compared to 20% (2/10) in the group of patients with an EGFR GCN,4.0 (Fishers Exact test, P = 0.0007). In the patient group treated with panitumumab +/2 cytotoxic therapy clinical benefit was 67% (8/12) in the patients with a high EGFR GCN and 44% (4/9) in those with a low EGFR GCN. The results are presented more in detail in Table S1.

Increased EGFR GCN Associates with Improved PFS and OS in Chemorefractory Disease
The median PFS of the chemorefractory patient cohort was significantly longer in the EGFR GCN$4.0 patients than in the EGFR GCN,4.0 patients; 29.5 vs. 10.8 weeks (Log-Rank test, P, 0.0001; Cox test, P,0.0001, HR = 0.23, 95% CI 0.12-0.46). The median OS time for patients with EGFR GCN$4.0 tumors was 12.5 months compared to 7.2 months for those with EGFR GCN below the cut-off value (Log-Rank test, P = 0.0002; Cox test, P = 0.0003, HR = 0.32, 95% CI 0.17-0.59). Kaplan-Meier survival curves are shown in Figure 1 c-d.
PFS remained longer in the patients with a high EGFR GCN regardless of which anti-EGFR mAb was used. In the patients treated with cetuximab +/2 cytotoxic therapy the median OS time was statistically significantly longer in the cohort of patients with an EGFR GCN$4.0 as compared to those with an EGFR GCN below 4.0 (12.5 vs. 4.6 months; Log-Rank test P = 0.0006; Cox test P = 0.001, HR 0.25, 95% CI 0.10-0.58), whereas no statistically significant OS difference was seen in the patients treated with panitumumab. The results are shown in Table S1.
The survival data of the original discovery-, the independent validation-, and combined chemorefractory patient cohorts are compared in Figure 2.

EGFR GCN Heterogeneity within Tumors
EGFR GCN was randomly evaluated, without IHC guidance, from all 31 validation cohort tumors. The mean EGFR GCN values from randomly selected areas were significantly lower when compared to the method where the EGFR GCN was evaluated selectively from areas with highest EGFR protein expression (P, 0.0001). The median EGFR GCN of these 31 primary CRC tumors was 4.3 when IHC guidance was used and 3.3 when the analysis was performed in a random fashion. The heterogeneity of EGFR GCN within one CRC tumor sample is demonstrated in Figure 3.
When the randomly chosen EGFR GCN values were used for survival analyses (EGFR GCN$4.0 vs. EGFR GCN,4.0) no statistically significant difference was observed between the two groups (PFS: P = 0.07, HR 0.40, 95% CI 0. 15

EGFR GCN and Response to Anti-EGFR Abs In vitro
We searched the Sanger Center cancer cell line database (http://www.sanger.ac.uk/cgi-bin/genetics/CGP/cghviewer/ CghHome.cgi) for EGFR GCN alterations. A low level of EGFR GCN increase (4-15 copies/cell), typically due to Chromosome 7 polysomy, was seen in 18 out of 39 (46%) cell lines. To correlate the EGFR GCN and KRAS status with anti-EGFR mAb response, we chose four lines for analysis. The CW-2 and C2BBe1 cell lines are KRAS-WT and have two and four copies of EGFR, respectively. NCI-H747 and SK-CO-1 are both KRAS mutant and have more than four copies (6-7) of EGFR. The EGFR GCN was confirmed by SISH (Figure 4a). The EGFR GCN was directly reflected in EGFR protein expression as indicated by Western blot analysis (Figure 4b). In cell viability assays, the KRAS-WT cell line with EGFR GCN 4 (C2BBe1) was the most sensitive to both cetuximab and panitumumab treatment (Figure 4c). The difference was highly significant when compared to any of the other cell lines (Student's t-test, P,0.001). Interestingly, the EGFR disomic, WT KRAS cell line (CW-2) was most resistant to mAb treatment. The cell lines with mutant KRAS and EGFR GCN.4 showed intermediate sensitivity, especially to panitumumab treatment. The results are thus in agreement with our clinical data indicating that both EGFR GCN and KRAS status define the tumor cell response to anti-EGFR mAbs.
We further looked at the effect of anti-EGFR mAb treatment on intracellular signaling in these cell lines. In KRAS mutant cell lines with more than four EGFR gene copies, phosphorylation of EGFR was evident and efficiently blocked by anti-EGFR mAb (Figure 4e). Anti-EGFR inhibition partially reduced pERK1/2 levels, while the level of pAKT was not affected. In KRAS-WT cell lines with 2-4 EGFR gene copies EGFR phosphorylation was under detection limit (not shown). However, the pathway was apparently operational as indicated by responses on downstream signaling. In the EGFR disomic line CW-2, anti-EGFR mAb treatment reduced pERK1/2 level, but did not affect AKT phosphorylation (Figure 4e). Only in EGFR polysomic and wild-type KRAS C2BBe1 cells, an effective blockage of both ERK1/2 and AKT signaling, was detected (Figure 4e). These results thereby suggest that both EGFR GCN and KRAS mutation status determine the effect of anti-EGFR mAb on EGFR downstream signaling and colorectal cancer cell survival.

Discussion
In this study, we demonstrate that heterogeneous EGFR GCN increase is a strong predictor of anti-EGFR treatment benefit in metastatic CRC. The results extend our previous findings of a single institute patient cohort to an independent validation cohort. In addition, they demonstrate the predictive value of EGFR GCN for anti-EGFR therapy efficacy in chemorefractory CRC patients, the most important patient group eligible for this treatment. The results further show that intra-tumoral EGFR GCN heterogeneity is common in CRC. We hypothesize that this previously unaccounted EGFR heterogeneity is a reason for the reported poor correlation between EGFR GCN analysis and efficacy of anti-EGFR therapy, and suggest an improved method for predictive EGFR GCN testing.
The patient material in our study included patients treated with anti-EGFR therapy both in an early and chemorefractory phase of metastatic CRC therapy. To control for the confounding effects of chemotherapy sensitivity when combined with anti-EGFR mAbs on our results, the predictive value of EGFR GCN on anti-EGFR treatment response was evaluated separately in the subgroup of patients treated in a chemorefractory phase (third line or more). The results obtained in the chemorefractory subgroup were similar to the results of the entire patient cohort, which supports our interpretation that a high EGFR GCN is indeed a predictor of favorable anti-EGFR treatment response.
Both cetuximab and panitumumab in the chemorefractory patient subgroup demonstrated improved PFS in EGFR GCN$ 4.0 patients. Overall survival and disease control rate was statistically improved for the cetuximab cohort, and numerically but not statistically in the small panitumumab cohort. The two therapeutic Abs are of different type, panitumumab being a fully human mAb, whereas cetuximab is a mouse-human chimera. In vitro assay demonstrated identical cytotoxicity for the tested CRC lines, but in vivo, other mechanisms, including antibody-dependent cytotoxicity (ADCC) may be operational. In this respect, cetuximab and panitumumab may be different and therefore, increased EGFR GCN could better indicate sensitivity to cetuximab treatment [11]. According to pre-clinical and clinical data certain cytotoxic therapies +/2 immunomodulating agents, including e.g. the GILF (gemcitabine, irinotecan, levofolinic, and 5-fluorouracil), GILFI (gemcitabine, irinotecan, levofolinic acid, fluorouracil, aldesleukin), and GOLFIG (gemcitabine, oxaliplatin, levofolinate, 5-fluorouracil, GM-CSF, aldesleukin) regimens, have the ability to effectively up-regulate EGFR expression on colon cancer cells, thereby enhancing the sensitivity of the colon cancer cells to cetuximab-mediated ADCC [12][13][14]. In our study, panitumumab was used as single therapy more often than   cetuximab, 42.9 vs 6.5% respectively, which at least in part can explain the difference observed in the efficacy results between the two antibodies. Both antibodies, when not administered as single treatment, were combined with irinotecan based chemotherapy regimens. In our study, oxaliplatin or single capecitabine were not combined to anti-EGFR therapy in the chemorefractory phase of the disease. Two patients were given cetuximab and panitumumab sequentially and therefore excluded from this study. Therefore, neither the combination cytotoxic drug nor the administration of both antibodies sequentially, explains the difference in the results obtained with the different anti-EGFR antibodies. Alternatively, our result may reflect a difference between treated patients. Cetuximab was more commonly used in the discovery cohort, which defined the test cut-off value, and panitumumab in the validation cohort with only chemorefractory patients. As both cohorts were retrospective, we cannot exclude differences in the patient populations, which could account for the differences in treatment results.
Tumor heterogeneity has been suggested to contribute to difficulties encountered in the validation of oncology biomarkers [15]. In the present work, the mean EGFR GCN values were significantly lower when analyzed in a random fashion as compared to the values obtained by choosing the cells with the highest GCN. Only the latter method was able to distinguish patients according to their anti-EGFR treatment response. This supports the view that therapeutic decision making based on scoring the dominant phenotype may be misleading [15]. Even a minor percentage of mutant alleles and gene expression profiles can be crucial for treatment response [16].
EGFR GCN heterogeneity is a well-established phenomenon in gliomas [17]. Although EGFR GCN heterogeneity in CRC may have been identified earlier, this finding has been generally disregarded, and not utilized as a parameter in diagnostic analyses. In some respects, the association between EGFR GCN and treatment response resembles the findings of another EGFR family member, Her2, in gastric cancer. Her2 is amplified or overexpressed in 7-34% of gastric cancers [18]. Unlike in breast cancer, Her2 expression in gastric cancer shows marked intra-tumoral heterogeneity, and therefore diagnostic test interpretation differs from breast cancer [19]. The current predictive diagnostics for trastuzumab treatment in gastric cancer is based on a combination of Her2 IHC and Her2 GCN analysis of areas with highest IHC staining. In biopsies, such areas may cover just 5% of the tumor [19]. Although there are parallels, the Her2-gastric cancer algorithm differs from EGFR GCN in CRC. In gastric cancer, the cut-off value is based on gene to chromosome ratio $2.0, although most recent recommendations suggest that Her2 GCN. 6.0 can be considered as positive. In CRC, the EGFR GCN increase is typically a result of chromosome 7 polysomy and therefore the gene to chromosome ratio is not informative. On the other hand chromosome 17 is rarely polyploid in gastric cancer, which indicates a different biological mechanism for EGFR and Her2 increase.
Tumor heterogeneity has been suggested to underlie resistance to targeted cancer therapeutics [15]. In this respect, the finding that a minor population of EGFR GCN high cells defines treatment response is unexpected. As the EGFR GCN low cells do not respond to treatment (the EGFR GCN low tumors are unresponsive), the most obvious outcome would be treatment failure, when the EGFR GCN low cells take over. This appears, however, not to be the case, therefore alternative explanations must be considered. One possibility is that the EGFR GCN high cells possess biological activities that determine the outcome of the entire tumor cell population. In such case, EGFR high cells may have yet undefined properties, such as paracrine effects on the major tumor cell population or features of cancer stem cells. There is evidence that, indeed, the transcriptome of the EGFR GCN high cells differs from EGFR low cells and that the EGFR pathway is associated with intestine stem cell properties [20,21]. At present, the mechanism by which EGFR GCN high cells dictate treatment response remains unknown.
EGFR signaling results in activation of intracellular pathways such as Ras/Raf/MAPK/ERK and PI3K/AKT, which regulate cell proliferation and survival. Anti-EGFR mAb:s are suggested to exert their anti-cancer effects by blocking these pathways [22]. Our cell line studies suggested a mechanism, by which the combination of EGFR GCN and KRAS mutation status can regulate the response to anti-EGFR treatment. EGFR inhibition with mAbs led to suppression of AKT pathway activity only in cells with increased EGFR GCN and KRAS-WT. Although EGFR phosphorylation was effectively blocked in KRAS mutant cells, AKT signaling was not suppressed, apparently due to constitutively active KRAS. Interestingly, in EGFR disomic and KRAS-WT cells, AKT signaling was active but not responding to anti-EGFR mAb suggesting that cells with normal EGFR GCN utilize alternative mechanisms to sustain AKT signaling. Similarly, the ERK1/2 phosphorylation was fully supressed only in cells with increased EGFR GCN and WT KRAS. In conclusion, this study indicates that the diagnostic algorithm combining EGFR IHC and EGFR SISH is a highly promising method for selecting CRC patients benefiting from anti-EGFR treatment. When combined with KRAS mutation testing, the ratio of responsive patients in the test positive group may almost double as compared to KRAS test alone. If further test validation using prospectively collected and randomized patient cohorts confirms the test results, these findings may have a profound effect on future selection of CRC patients for anti-EGFR therapy.

Supporting Information
Table S1 Tumor response and survival of patients with chemorefractory KRAS wild type metastatic or locally advanced colorectal cancer treated with cetuximab or panitumumab according to EGFR Gene Copy Number. (XLS)


extfieldsofstudy: Medicine Biology

text:
The stem cell organisation, and the proliferative and gene expression profile of Barrett's epithelium, replicates pyloric-type gastric glands

Objective Barrett's oesophagus shows appearances described as ‘intestinal metaplasia’, in structures called ‘crypts’ but do not typically display crypt architecture. Here, we investigate their relationship to gastric glands. Methods Cell proliferation and migration within Barrett's glands was assessed by Ki67 and iododeoxyuridine (IdU) labelling. Expression of mucin core proteins (MUC), trefoil family factor (TFF) peptides and LGR5 mRNA was determined by immunohistochemistry or by in situ hybridisation, and clonality was elucidated using mitochondrial DNA (mtDNA) mutations combined with mucin histochemistry. Results Proliferation predominantly occurs in the middle of Barrett's glands, diminishing towards the surface and the base: IdU dynamics demonstrate bidirectional migration, similar to gastric glands. Distribution of MUC5AC, TFF1, MUC6 and TFF2 in Barrett's mirrors pyloric glands and is preserved in Barrett's dysplasia. MUC2-positive goblet cells are localised above the neck in Barrett's glands, and TFF3 is concentrated in the same region. LGR5 mRNA is detected in the middle of Barrett's glands suggesting a stem cell niche in this locale, similar to that in the gastric pylorus, and distinct from gastric intestinal metaplasia. Gastric and intestinal cell lineages within Barrett's glands are clonal, indicating derivation from a single stem cell. Conclusions Barrett's shows the proliferative and stem cell architecture, and pattern of gene expression of pyloric gastric glands, maintained by stem cells showing gastric and intestinal differentiation: neutral drift may suggest that intestinal differentiation advances with time, a concept critical for the understanding of the origin and development of Barrett's oesophagus.

ABSTRACT
Objective Barrett's oesophagus shows appearances described as 'intestinal metaplasia', in structures called 'crypts' but do not typically display crypt architecture. Here, we investigate their relationship to gastric glands. Methods Cell proliferation and migration within Barrett's glands was assessed by Ki67 and iododeoxyuridine (IdU) labelling. Expression of mucin core proteins (MUC), trefoil family factor (TFF) peptides and LGR5 mRNA was determined by immunohistochemistry or by in situ hybridisation, and clonality was elucidated using mitochondrial DNA (mtDNA) mutations combined with mucin histochemistry. Results Proliferation predominantly occurs in the middle of Barrett's glands, diminishing towards the surface and the base: IdU dynamics demonstrate bidirectional migration, similar to gastric glands. Distribution of MUC5AC, TFF1, MUC6 and TFF2 in Barrett's mirrors pyloric glands and is preserved in Barrett's dysplasia. MUC2-positive goblet cells are localised above the neck in Barrett's glands, and TFF3 is concentrated in the same region.
LGR5 mRNA is detected in the middle of Barrett's glands suggesting a stem cell niche in this locale, similar to that in the gastric pylorus, and distinct from gastric intestinal metaplasia. Gastric and intestinal cell lineages within Barrett's glands are clonal, indicating derivation from a single stem cell. Conclusions Barrett's shows the proliferative and stem cell architecture, and pattern of gene expression of pyloric gastric glands, maintained by stem cells showing gastric and intestinal differentiation: neutral drift may suggest that intestinal differentiation advances with time, a concept critical for the understanding of the origin and development of Barrett's oesophagus.

INTRODUCTION
Barrett's oesophagus remains an enigma with no agreement about its origin, [1][2][3] and the nature of the epithelium in Barrett's mucosa is disputed, 4 5 with controversy regarding which epithelial component progresses to cancer. The belief that intestinal metaplasia (or 'specialised epithelium'), where goblet cells are plentiful, is typically required for the diagnosis of Barrett's oesophagus 2 has lead to Barrett's mucosa being regarded as 'intestinal metaplasia', and the mucosal units as 'crypts', resembling crypts in the intestine. However, the phenotype of Barrett's oesophagus is protean containing a variety of cell lineages. Even in 'specialised epithelium', there are multiple cell lineages: columnar cells Open Access Scan to access more free content

Significance of this study
What is already known on this subject?
▸ Barrett's glands are protean and contain a wide range of differentiated cell lineages. ▸ Barrett's glands are clonal and contain multiple, multipotent stem cells. ▸ Dogma states that Barrett's is a metaplasia of the squamous oesophagus.
What are the new findings?
▸ The gene expression and proliferative compartments of Barrett's glands reflects that of pyloric glands which also demonstrate bidirectional migration, as seen in gastric glands. ▸ The stem cell zone is located at the neck of Barrett's glands, similar to pyloric glands: gastric and intestinal cell lineages within Barrett's glands show a common stem cell origin. ▸ 'Specialised' gastric glands resemble pyloric glands showing partial intestinalisation. ▸ The gastric gland architecture and organisation is maintained in dysplasia.
How might it impact on clinical practice in the foreseeable future?
▸ Endoscopic and pathological examination of the gastric cardia may need to be more rigorous in gastro-oesophageal reflux disease patients. ▸ More investigation into determining glandular phenotype as a biomarker of progression to dysplasia in Barrett's patients is needed. ▸ Any unifying proposal for the origin and development of Barrett's oesophagus should explain their resemblance to pyloric glands.
resembling gastric foveolar cells containing MUC1, MUC5AC and mucus secreting cells expressing MUC6-mucin core proteins characteristic of gastric epithelium, 6 7 and goblet cells, with MUC2 and MUC3-seen in intestinal epithelium. 8 Thus, the so-called 'specialised epithelium' of Barrett's oesophagus, often compared with intestinal metaplasia, shows evidence of gastric lineage differentiation as well as intestinal differentiation. Barrett's mucosa contains several different types of glands-Paull et al, 9 by mapping the distribution of the several phenotypes, reported a zonal distribution of the different types of mucosa, with oxyntic-type glands with parietal and chief cells or oxynto-cardiac glands interposed between the specialised columnar epithelium and the lower oesophageal sphincter. Such 'zonation' has been replicated, although some reports 10 11 have found the different phenotypes randomly distributed throughout Barrett's mucosa. There is a gradient of goblet cell density, with significantly lower numbers seen in the distal Barrett's segment, 10 correlated with an oesophageal luminal pH gradient. 11 Cardiac mucosa is present throughout the segment, with oxynto-cardiac mucosa more frequently found distally. 9 10 Going et al, 10 reported higher frequencies of cardiac and oxynto-cardiac mucosa in the distal Barrett's segment, with several different mucosal phenotypes at any single anatomical level, although 'specialised' epithelium was found at all levels. Glands showing intestinalisation, resembling 'complete type' (Type I) intestinal metaplasia, characterised by the presence of absorptive cells, Paneth cells, and goblet cells, are also seen. 10 Thus, Barrett's oesophagus contains multiple lineages and glands with several phenotypes.
Human intestinal crypts and gastric glands are clonal populations-derived from a single tissue-specific stem cell. 12 These clonal crypts and glands contain multiple, multipotential stem cells from which all the contained lineages derive: studies using mitochondrial DNA (mtDNA) mutations that cause cytochrome c oxidase (CCO) deficiency as clonal markers, showed Barrett's metaplastic glands as clonal units maintained by multiple stem cells, and all epithelial cell lineages within a gland derived from multipotential stem cells. 13 Thus, whatever the complexity of a Barrett's gland, whatever heterogeneous cell lineages it contains, it was derived from a single stem cell and that original stem cell's progeny has sufficient multipotentiality to maintain its multilineage habitus.
Barrett's glands show evidence of gastric and intestinal differentiation patterns: if such glands are clonal, the stem cell(s) will show capacity to differentiate into gastric and intestinal cell lineages. We show that specialised Barrett's glands show maximal proliferation in the middle part of the gland, that cells migrate in a bidirectional manner and that the stem cell niche is located in the middle part of the gland, resembling the gastric gland and not the intestinal crypt. Region-specific gene expression supports a gastric gland plan, and we propose that Barrett's glands are maintained by stem cells with gastric and intestinal differentiation capacity that progress to intestinal type over time.

MATERIALS AND METHODS
Tissues: formalin-fixed, paraffin-embedded archival nondysplastic Barrett's oesophagus and Barrett's dysplasia tissue specimens and frozen specimens were obtained from patients undergoing oesophagectomy or endoscopic mucosal resection for adenocarcinoma or dysplasia (n=34). Normal gastric and intestinal metaplastic formalin-fixed, paraffin-embedded specimens were obtained from patients undergoing resection for either gastric carcinoma or high-grade dysplasia (n=23).
Histological examination following standard H&E staining and periodic acid Schiff/Alcian blue staining was carried out by at least two qualified pathologists (RH, MR-J, MRN, NS or NAW). Ethical approval was sought and obtained from the London research ethics committee, Stanmore11/LO/1613. Immunohistochemistry (IHC) was carried out using methods described in online supplementary methods. The numbers of Ki67+ and IdU+ cells were scored within Barrett's glands as follows: two tissue sections from each of the patients were included and three areas of approximately 100 cells were scored per section. For cell counts, glands were divided into three equal regions: the bottom third was designated the gland basecorresponding to the Muc6+/trefoil family factor 2 (TFF2)+ mucus secreting zone, and the remaining upper two-thirds of the gland were divided equally and designated the middle region and the surface of the gland, respectively (highlighted in figure 1A).
In situ hybridisation (ISH) was carried out using the methods described in online supplementary methods.
Clinical protocol for labelling with iododeoxyuridine (IdU): The Stem Cell Assessment In Neoplastic Tissues (SAINT) trial (n=4 patients) was approved by the Leicestershire Ethics Board Ref Number: 09122, Medicines Health Regulatory Authority Number: CTA 21275 and Research Ethics Committee Number: 7213 as previously described. 14 Prior to oesophagectomy, oesophageal Barrett's adenocarcinoma patients were infused with IdU, and details are given in online supplementary methods.
Laser capture microdissection and PCR sequencing of MtDNA: This was carried out using the methods described in online supplementary methods.

Proliferative organisation in Barrett's glands
Ki67+ cells were concentrated in the middle region of Barrett's gland with fewer proliferating cells at the surface of the glands, little at the bases and none in the very basal cells (figure 1A, and online supplementary figure S1). This pattern of expression was consistent throughout all study patients. The majority of Ki67+ cells were seen within the middle region of the gland in all patients (54.5% of total cells). The percentage of Ki67+ cells within the middle region was shown to be significantly greater than that of the base of the gland and the gland surface within these patients (Kruskal-Wallis one-way analysis of variance, p<0.05).

Cell migration in Barrett's glands
At 7 days postinjection, IdU+ cells were seen towards the base and middle of the gland and also at the surface (figure 1B), clustered together in these gland segments. At 11 days, IdU+ cells were seen in the base of the gland, in the middle and also at the surface of the gland, but the number of IdU+ cells was reduced compared to 7 days with the majority of positive cells seen towards the gland bases (figure 1B) suggesting that the superficial labelled cells have been rapidly lost into the lumen, with only a few still migrating, but that migration towards the base is slower. At 29 days after injection, IdU+ cells were still evident, although greatly reduced compared to 11 days, and were seen almost exclusively in the bases of the glands, where a few cells were still evident at 67 days after injection 1 . 4 The distribution of IdU+ cells within Barrett's glands over time was quantified as described above: figure 1C shows the distribution of IdU+ cells in Barrett's glands with time after IdU injection. After 7 days, 4.45% of IdU+ cells were observed within the surface of the gland, although labelled cells were still numerous within the middle region (3.65%). However, fewer IdU+ cells were observed within the gland base (1.74%). After 11 days, IdU+ cells were still observed within the surface of the glands, although the fraction had significantly reduced and they now represented 0.07% of total cells. The highest percentage was observed within the base of the Barrett's gland (1.36%). After 29 days chase, the number of IdU+ cells was significantly reduced compared to 11 days, and were exclusively observed within the base of the Barrett's glands (0.181% of total cells). Furthermore, the number was reduced again after 67 days chase: at this time IdU+ cells were seen exclusively within the base of the gland (0.07%).
Taken together with the Ki67 labelling index distribution, which shows that the bases of Barrett's glands contain few labelled cells (see online supplementary figure S1), and since maximal cell proliferation is present in the middle of the gland, cells migrate faster towards the surface or foveolar portion of the gland, and slowly into the base of the gland. Labelled cells in Barrett's glands show bidirectional flux.
In corpus gastric glands, one tissue section from each patient was included and three areas of over 100 cells within each region of the gastric unit (foveolus or pit, isthmus/neck and gland base) and were scored per slide. Online supplementary figures S2A,B show that Ki67-labelled cells occur mainly within the neck/isthmus regions of the gastric unit (15.2% of total cells) with fewer Ki67+ cells observed within the foveolus (9.5%) and 1.5% in the gland base. A similar distribution is shown in gastric antral mucosa (see online supplementary figure S2B). After 7 days, the highest percentage of IdU+ cells was observed within the foveolar region (3.3%) (see online supplementary figures S2C,D), whereas at 11 days, the highest percentage of cells was observed within the neck (0.27%) (see online supplementary figures S2E,F). There was a significant reduction in IdU+ cells within the neck and the foveolar regions between 7 and 11 days, suggesting that cellular flux occurs mainly in the foveolus, since 7 days postinjection most IdU+ cells are identified within this region. Most cells are lost into the lumen after 11 days, yet some cells remain within the neck region of the gland. However, IdU-labelled parietal cells were seen towards the bases of gastric glands at 67 days after infusion of IdU 14 .
Cell flux is bidirectional in Barrett's glands, similar to that seen in gastric glands.

The stem cell niche in Barrett's glands
Here, stem cells were identified by ISH for the established gastrointestinal epithelial stem cell marker LGR5. 15 Figure 2 shows the distribution of cells which express LGR5 mRNA in Barrett's glands (A, B), in pyloric glands (C, D) and in the crypts of gastric intestinal metaplasia (C, F). Figures are representative of n=5. In the pyloric glands ( figure 2C,D) LGR5 mRNA is seen quite widely distributed in the isthmus/neck area of the glands, while the foveola and the mucin-secreting bases of the glands are negative. In Barrett's glands ( figure 2A,B) LGR5 mRNA is localised in the middle of the gland, corresponding to the equivalent of the isthmus/pit in a pyloric gland. Figures 2E and F show that in intestinal metaplasia in the stomach, LGR5 mRNA is found at the bases of the crypts, similar to colonic crypts (see online supplementary figure S3).
The stem cell niche in Barrett's oesophagus is localised in the middle of the gland, in the equivalent of the isthmus/pit of a pyloric gland, as seen in pyloric gastric glands. In gastric intestinal metaplasia, the stem cell niche is at the base of the crypt, comparable with the colon. Gene expression in Barrett's glands Figure 4Ai shows that appropriately sectioned Barrett's glands appear slender and elegant, with a single surface opening or foveolus, and about half-way down the gland divides into a number of basal tubules, similar to the disposition of the gastric gland. 12 The upper part of the gland contains columnar cells that stain with D/PAS and Alcian Blue, and there are also numbers of alcianophilic, sialomucin-containing goblet cells and non-goblet cells. In the base of the gland, there is an area, of variable size, which contains D/PAS+ mucous cells only. The proliferative zone (Ki67+) in this gland is seen above this area, extending into the upper part of the gland (figure 4Aii), similar to that seen in gastric glands (figure 4Aiii).

Clonal organisation of Barrett's glands
In Barrett's glands and pyloric glands, MUC5AC is seen exclusively in the upper part of the gland, decreasing in intensity as the stem cell zone, delineated by LGR5 mRNA expression, is reached (figure 4Aiv and Bi respectively). MUC6 expression in Barrett's and pyloric glands is confined to the mucous cells at the base of the glands below the LGR5 mRNA zone (figure 4Bii and iii). MUC2 shows a tight distribution throughout the upper part of the Barrett's gland, concentrated but not confined to the goblet cells, diminishes in expression towards the LGR5 zone, and is absent from the MUC6+ bases (figure 4Biv). MUC2 is absent from the pyloric epithelium (see online supplementary figure S5A). TFF1 protein and mRNA is located in the upper part of Barrett's and pyloric glands, coexpressed with MUC5AC (figure 4Ci, ii and iii, respectively, and see online supplementary figure S5B). TFF2 is seen confined largely to the MUC6+ cells in the base of Barrett's and pyloric glands (figure 4Civ, Di and ii, respectively, and see online supplementary figure S5C). TFF2 mRNA is also present in low concentration in the foveolar equivalent of the Barrett's gland (figure 4Di, arrow). TFF3 protein is expressed throughout the Barrett's gland, concentrated in the goblet cells in the upper part of the gland (figure 4Diii), confirmed by the distribution of TFF3 mRNA (figure 4Div). TFF3 is absent from the pyloric mucosa, unless intestinal metaplasia is present (see online supplementary figure 5Di and ii). In total, 15 patients with Barrett's metaplasia and 15 normal stomachs taken outside the resection margins of patients with gastric adenocarcinoma were used. All results described were observed in all samples.
The pattern of gene expression in antral or pyloric mucosa is thus reflected in Barrett's glands. H&E): the proliferative organisation mirrors that seen in Barrett's glands and, thus, in gastric glands, with preservation of the non-proliferative MUC6+/TFF2+ cells at the base of the gland. Figure 5B shows that the proliferative zone indicated by Ki67 expression expands towards the top of the glands, while the bases remain largely non-proliferative. MUC2 is present in the middle and top of the glands (figure 5C) and MUC5AC on the surface and in the middle of glands (figure 5D): the basal TFF2+/MUC6+ mucous zone is preserved ( figure 5E and F).

Cell proliferation and gene expression in Barrett's dysplasia
LGR5 mRNA+ cells are seen again above the MUC6+/TFF2+ cells, but there is some expansion of the zone towards the surface ( figure 6Ai-Cii). These data are representative of 15 Barrett's dysplasia specimens. In Barrett's carcinoma ( figure 6Di  and ii) LGR5 mRNA shows a specific localisation at the bases of the malignant glands (n=5).

Gene expression in gastric intestinal metaplasia (IM)
In gastric intestinal metaplasia, especially in the so-called 'mixed', there is expression of gastric and intestinal mucins. 16 Similarly, in partially intestinalised gastric glands, the distribution of the TTFs follows the pattern seen in Barrett's glands (see online supplementary figure S6). TFF2 and MUC6 (see online supplementary figure 6A, D) is seen at the bases, in continuity with goblet cell-containing intestinalised glands. TFF1 mRNA is seen at the apices of such partially intestinalised glands (see online supplementary figure S6B), even where goblet cell differentiation is clearly seen (arrow). TFF2 mRNA is concentrated in the gland base (see online supplementary figure S6C), although as in Barrett's glands (figure 4, white arrow), it is also seen in the goblet cell-containing surface (black arrow). TFF3 mRNA is seen throughout the gland ( figure 6E), but prominent in surface goblet cells (arrow).
LGR5 mRNA expression becomes localised in the isthmus/neck equivalent in dysplastic glands (see online supplementary figure S6Fi and ii), and the basal MUC6+/TFF2+ zone is preserved (arrowed).

DISCUSSION
The gland pattern of Barrett's glands is that of glands of the pyloric mucosa: the proliferative zone is housed within the isthmus/neck equivalent of the gland, and cell migration is bidirectional, upwards into the foveolus equivalent, and downwards into the tubules of the gland base. The stem cell zone, as indicated by the presence of LGR5 mRNA+ cells, is located in the lower part of this area, almost at the level of the D/PAS+, TFF2+,

MUC6+ cells (figure 4), while
LGR5 mRNA+ cells in the antral glands are seen mainly in the isthmus/neck. MUC5AC and TFF1 are found in the upper part of the gland, while TFF2 and MUC6 concentrated in the D/PAS cells at the gland base, as seen in antral gastric glands. MUC2, and to a lesser extent TFF3, is seen predominantly in goblet cells in the upper part of the gland. The organisation of Barrett's glands, their proliferative architecture, stem cell localisation and patterns of gene expression, directly reflects the pyloric gastric gland. We conclude that Barrett's glands contain equivalents of the foveolus, isthmus, neck and base seen in gastric glands.
The distribution of Ki67+ cells has been studied in Barrett's glands previously, and similar patterns seen to those described herein, persist even in dysplasia. 17 The dynamic aspects of cell migration have not been studied: it should be recalled that Ki67 labels all cells in the cell cycle and the distribution seen in figure 1 shows MUC5AC protein also in the upper part of pyloric glands; (iv) TTF2 protein in Barrett's glands. Figure 4D (i) mRNA expression in Barrett's glands; (ii) TFF2 mRNA in pyloric glands; (iii) TFF3 protein and (iv) mRNA in Barrett's glands.
reflects the distribution of cycling cells, effectively at time 0. IdU labels cells only in the S phase, and earlier readings available at 7 days would then reflect the position of cells labelled 7 days previously (note that the very basal gland is devoid of Ki67+ cells: see online supplementary figure S1). The fraction of IdU+ cells labelled in the foveolar region now equals that seen in the erstwhile proliferative zone. This means that cells have migrated, fairly rapidly, upwards to the foveolus. In the base, there are, at this time, relatively few labelled cells, indicating that migration to the base is slower. At 11 and 29 days, while labelled cells are lost from the foveolar region (by extrusion from the surface), and from the proliferative zone, labelled cells remain in the base, and by 67 days, only the base contains any labelled cells.
Migration in Barrett's glands is bidirectional: the detailed studies of Hattori and Fujita 18 in the hamster corpus and pylorus have shown that, while cells migrate to the surface in some 14 days, over 300 days are needed to reach the corpus gland base. In humans, foveolar cells migrate to the surface rapidly (see online supplementary figure S1), but labelled parietal cells are seen in the isthmus at 67 days after IdU injection, 14 indicating that in the human being, a similar lengthy period is needed for cells to reach the gland base, confirming bidirectional flux.
LGR5 has been shown to be a true stem cell marker in the mouse by lineage labelling. 15 LGR5+ cells represent a stem-like cell population in colon carcinomas, which are also found in the bases of crypt-like structures within the tumour that resembles normal crypts 19 (and see figure 6D). The presence of LGR5+ cells and all differentiated lineages within colon carcinomas 20 and adenomas has been reported, 19 suggesting that LGR5 detects stem cell populations in human epithelia.
LGR5 mRNA localises specifically to the junction between the TFF1 +/MUC5AC+/MUC2+ cells and the basal TFF2+/MUC6+ cells, and represents the stem cell zone, or niche, of Barrett's glands. Cells, including the MUC2+ cells destined to be goblet cells, migrate upwards from here, while the TFF2+/MUC6+ cells migrate downwards. In pyloric glands, LGR5 is also localised in the isthmus/neck region, and a similar pattern of gene expression is seen. Contrast these findings with those exhibited in gastric intestinal metaplasia (figure 2E,F): LGR5 mRNA is seen in the base, as in the small intestinal crypt (see online supplementary figure S3) and colonic crypts. 19 Barrett's carcinoma glands show LGR5 mRNA localisation very similar to that described by Merlos-Suarez et al 20 (ISH was used as IHC for human LGR5 is unreliable) ( figure 6D).
There have been previous studies of the distribution of mucin gene expression 6 21 and also of TFFs 22 23 in Barrett's glands: selective segregation of MUC5AC with TFF1 and MUC6 with TFF2 is seen in Barrett's glands, mirroring antral gastric glands. 24 TFF3 is usually colocalised with MUC2, but in Barrett's glands, it is expressed in the MUC6+/TFF2+ basal mucous cells, differing from antral glands and also in incompletely intestinalised intestinal metaplastic glands (see online supplementary figure S6E). TFF3 expression has been described in the normal human stomach, 25 although in our hands not unless intestinal metaplasia is present (see online supplementary figure S5D). TFF2 mRNA is also expressed, in low concentration, in the 'foveola' of Barrett's and partially intestinalised gastric glands (see figures 4Di, ii and also online supplementary figure S6C).
An intriguing point is the relationship of Barrett's metaplasia to intestinal metaplasia. 26 'Mixed' intestinal metaplasia occurs in the stomach, 16 and online supplementary figure S6 shows that such mixed types of intestinal metaplasia show a similar pattern of TFF expression as Barrett's glands-the mucin histochemical profiles also show a mixed pattern, 16 suggesting that Barrett's glands resemble the mixed metaplastic glands seen in the stomach.
Barrett's glands are clonal populations 13 (figure 3). Established Barrett's glands contain multiple cell lineages, MUC5AC+/TFF1+, MUC2+ and MUC6+/TFF2+, and in Barrett's epithelium, gastric and intestinal endocrine cells are found. 27 The stem cell niche in the isthmus/neck equivalent area of the gland contains stem cells capable of delivering all these lineages. Either the niche contains unique stem cells capable of giving rise to all contained lineages, or the niche is composed of a mixture of stem cells with limited repertoire, for example, limited to TFF1+/MUC5AC+ and TFF2+/MUC6+ lineages, and other stem cells give rise to the TFF3+/MUC2+ lineage ( figure 7).
The implication here is that Barrett's glands were originally gastric glands containing TFF1+/MUC5AC+ and TFF2 +/MUC6+ lineages. The homeobox gene, CDX2, is associated with activation of intestinal differentiation. 2 This occurs in the stomach as a result of Helicobacter pylori infection, and could occur in Barrett's glands as a result of continued bile/acid Figure 6 LGR5 mRNA expression in Barrett's dysplasia and carcinoma. (A-C) low-(i) and high-power (ii) images showing non-isotopic ISH for LGR5 mRNA localisation in Barrett's dysplasia; (D) (i) bright field image and accompanying dark field image (ii) of isotopic ISH for LGR5 mRNA localisation in invasive Barrett's carcinoma glands.
refluxate. CDX2 expression in a stem cell will induce progenitors of the TFF3+/MUC2+ lineage. Most stem cell divisions are symmetrical, and neutral drift governs stem cell dynamics: 28 stochastically, a TFF3+/MUC2+ stem cells may survive in the niche, and the gland then contains the first stem cell committed to intestinal differentiation. Neutral drift dynamics would predict that such a stem cell could be lost, and the gland maintains its gastric phenotype, but the stem cell could expand in the niche, giving the Barrett's phenotype of mixed gastric and intestinal lineages. Eventually, niche succession would fill the niche with MUC2 +/TFF3+ stem cells, giving a fully intestinalised gland, seen in Barrett's oesophagus. 9 10 We would state that causality cannot be inferred from the observational data provided. Gastric intestinal metaplasia may also go through such a transitional phase 16 (see online supplementary figure S6). Cells expressing gastric and intestinal markers are seen, possibly the progeny of stem/progenitor cells where CDX2 is only partially activated, and where gastric and intestinal gene expression coexists.
The gland pattern, including the basal TFF2+/MUC6+ cells, are preserved in dysplasia, with dysplastic cells confined to the upper part of the gland; in gastric carcinoma, the dysplastic cells may originate in the deep pit or isthmus of the gastric gland: 29 in Barrett's oesophagus, the dysplastic part of the gland is clonal for p16 mutations even when the surface cells lack dysplastic features. 30 It is not yet known if this is true also for the basal TFF2+/MUC6+ basal mucous cells. Dysplasia and indeed carcinoma can show gastric and intestinal differentiation markers. 7 Dysplasia may arise through mutational events in stem cells: hence, dysplasia developing in TFF1+/MUC5AC+ or in TTF3 +/MUC2+ committed stem/progenitor cells may account for the origin of the dysplasia in the stem cell zone and also for the several differentiation patterns described. 7 Barrett's segments may develop from upward progression of the cardiac mucosa, a concept which appears to have fallen out of favour in recent years in favour of a metaplastic origin from oesophageal squamous epithelium. 2 But there is older evidence, such as the development of neo-Barrett's after subtotal oesophagectomy and reconstruction with a gastric conduit: about 50% of these patients develop columnar epithelium in the area lined by squamous mucosa at the time of the procedure, and the length of columnar mucosa increases with longer follow-up. 31 Recent evidence from animal models, 1 and from following the progression of metaplasia at the cardia, 32 is supportive of such a proposal. Whichever theory is entertained, it will have to account for the observation that Barrett's glands replicate the organisation of gastric glands. following an event such as activation of CDX2, this stem cell(s) commit to produce TFF3+/MUC2+ cells, and stochastic niche succession will eventually, in some glands, move entirely to a niche containing stem cells committed to the TFF3+/MUC2+ lineage. We propose a conversion from non-goblet containing columnar to a specialised epithelium and finally to intestinal metaplasia.