Durga Attili

Human colonic crypts in culture: segregation of immunochemical markers in normal versus adenoma-derived

In order to advance a culture model of human colonic neoplasia, we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomas. Crypts were maintained in three-dimensional Matrigel culture with a simple, serum-free, low Ca2+ (0.15 mM) medium. Intact colonic crypts from normal human mucosa were viably maintained for 3–5 days with preservation of the in situ crypt-like architecture, presenting a distinct base and apex. Abnormal structures from adenoma tissue could be maintained through multiple passages (up to months), with expanding buds/tubules. Immunohistochemical markers for intestinal stem cells (Lgr5), growth (Ki67), differentiation (E-cadherin, cytokeratin 20 (CK20) and mucin 2 (MUC2)) and epithelial turnover (Bax, cleaved Caspase-3), paralleled the changes in function. The epithelial cells in normal crypts followed the physiological sequence of progression from proliferation to differentiation to dissolution in a spatially and temporally appropriate manner. Lgr5 expression was seen in a few basal cells of freshly isolated crypts, but was not detected after 1–3 days in culture. After 24 h in culture, crypts from normal colonic tissue continued to show strong Ki67 and MUC2 expression at the crypt base, with a gradual decrease over time such that by days 3–4 Ki67 was not expressed. The differentiation marker CK20 increased over the same period, eventually becoming intense throughout the whole crypt. In adenoma-derived structures, expression of markers for all stages of progression persisted for the entire time in culture. Lgr5 showed expression in a few select cells after months in culture. Ki67 and MUC2 were largely associated with the proliferative budding regions while CK20 was localized to the parent structure. This ex vivo culture model of normal and adenomatous crypts provides a readily accessible tool to help understand the growth and differentiation process in human colonic epithelium.

Iron Uptake via DMT1 Integrates Cell Cycle with JAK-STAT3 Signaling to Promote Colorectal Tumorigenesis.

Dietary iron intake and systemic iron balance are implicated in colorectal cancer (CRC) development, but the means by which iron contributes to CRC are unclear. Gene expression and functional studies demonstrated that the cellular iron importer, divalent metal transporter 1 (DMT1), is highly expressed in CRC through hypoxia-inducible factor 2α-dependent transcription. Colon-specific Dmt1 disruption resulted in a tumor-selective inhibitory effect of proliferation in mouse colon tumor models. Proteomic and genomic analyses identified an iron-regulated signaling axis mediated by cyclin-dependent kinase 1 (CDK1), JAK1, and STAT3 in CRC progression. A pharmacological inhibitor of DMT1 antagonized the ability of iron to promote tumor growth in a CRC mouse model and a patient-derived CRC enteroid orthotopic model. Our studies implicate a growth-promoting signaling network instigated by elevated intracellular iron levels in tumorigenesis, offering molecular insights into how a key dietary component may contribute to CRC.

Identification, isolation, and characterization of human LGR5-positive colon adenoma cells

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis. Summary: Immunohistochemical and transcriptomic analyses of organoids generated from precancerous adenoma, colon adenocarcinoma and normal human tissue shows that the intestinal stem cell marker LGR5 is a colon cancer prognostic factor.

Monoclonal antibodies specific for oncofetal antigen – immature laminin receptor protein: Effects on tumor growth and spread in two murine models

The oncofetal antigen – immature laminin receptor protein (OFA/iLRP) has been linked to metastatic tumor spread for several years. The present study, in which 2 highly-specific, high-affinity OFA/iLRP-reactive mouse monoclonal antibodies were examined for ability to suppress tumor cell growth and metastatic spread in the A20 B-cell leukemia model and the B16 melanoma model, provides the first direct evidence that targeting OFA/iLRP with exogenous antibodies can have therapeutic benefit. While the antibodies were modestly effective at preventing tumor growth at the primary injection site, both antibodies strongly suppressed end-organ tumor formation following intravenous tumor cell injection. Capacity of anti-OFA/iLRP antibodies to suppress tumor spread through the blood in the leukemia model suggests their use as a therapy for individuals with leukemic disease (either for patients in remission or even as part of an induction therapy). The results also suggest use against metastatic spread with solid tumors.

A Method for Cryogenic Preservation of Human Biopsy Specimens and Subsequent Organoid Culture

Human tissue–derived gastrointestinal (GI) organoids have revolutionized the study of human biology, and are powerful tools for studying human physiology and disease; however, generation of organoids is limited by access to human tissue and a short window of viability for human samples, putting a hard limit on the time and place in which a patient sample can be used for research. These restraints mean that a laboratory must be relatively geographically close to the source of collection to use the sample within the window of viability. Patient-derived organoids also are being used for drug development, stem cell therapies, and personalized medicine; however, it is not always feasible to prospectively develop organoid line efforts given the time and labor involved. To overcome these limitations, we sought to develop a practical method to cryopreserve live human biopsy tissue, which then could be stored or shipped frozen and later thawed to generate new cultures of GI epithelium-only organoids (also referred to as enteroids/colonoids). Here, we describe a simple and robust method to cryopreserve human biopsy specimens that subsequently could be

Calcium - Induced Differentiation of Human Colon Adenomas In Colonoid Culture: Calcium alone versus calcium with additional trace elements

Previous murine studies have demonstrated that dietary Aquamin, a calcium-rich, multi-mineral natural product, suppressed colon polyp formation and transition to invasive tumors more effectively than calcium alone when provided over the lifespan of the animals. In the current study, we compared calcium alone to Aquamin for modulation of growth and differentiation in human colon adenomas in colonoid culture. Colonoids established from normal colonic tissue were examined in parallel. Both calcium alone at 1.5 mmol/L and Aquamin (provided at 1.5 mmol/L calcium) fostered differentiation in the adenoma colonoid cultures as compared with control (calcium at 0.15 mmol/L). When Aquamin was provided at an amount delivering 0.15 mmol/L calcium, adenoma differentiation also occurred, but was not as complete. Characteristic of colonoids undergoing differentiation was a reduction in the number of small, highly proliferative buds and their replacement by fewer but larger buds with smoother surface. Proliferation marker (Ki67) expression was reduced and markers of differentiation (CK20 and occludin) were increased along with E-cadherin translocalization to the cell surface. Additional proteins associated with differentiation/growth control [including histone-1 family members, certain keratins, NF2 (merlin), olfactomedin-4 and metallothioneins] were altered as assessed by proteomics. Immunohistologic expression of NF2 was higher with Aquamin as compared with calcium at either concentration. These findings support the conclusions that (i) calcium (1.5 mmol/L) has the capacity to modulate growth and differentiation in large human colon adenomas and (ii) Aquamin delivering 0.15 mmol/L calcium has effects on proliferation and differentiation not observed when calcium is used alone at this concentration. Cancer Prev Res; 11(7); 413–28. ©2018 AACR.

Abstract 322: Establishment and genomic characterization of enteroid cultures from human colonic adenomas and adenocarcinomas

Introduction Characterized enteroid cultures of human colon cancer can more precisely model the diversity of colonic neoplasia for the study of cancer initiation, progression and potentially prevention. Using tissue from colon resections and endoscopic biopsies, we have successfully isolated and cultured 14 colorectal adenomas and 2 adenocarcinomas to date. We have maintained these enteroid cultures for up to 2 years and established a working cryorepository. Specific epithelial cell lineage markers and the stem cell marker Lgr5 can be detected throughout the culture period. Methods Enteroid cultures have been initiated and maintained in a serum-free medium containing EGF and pituitary extract. However, approximately half of all neoplasms do not establish in this reduced medium. In contrast, most neoplasms develop and expand in an enriched culture medium containing serum, Wnt, R-spondin, Noggin, and EGF. We have also created a mouse xenograft from an adenoma expanded in the reduced media; this graft was then successfully reintroduced into culture. Using whole exome sequencing, we are investigating how the genetic background of individual patients contributes to 1) variability in the establishment and expansion of enteroid cultures 2) tumor heterogeneity in neoplasms and xenografts, and 3) the stability of genomic signatures in enteroids over time in culture. Results Twenty-two damaging somatic variants identified in a single colon tumor were preserved in enteroid culture after 2 months in reduced medium. Variants included a frameshift mutation in APC and missense mutations in KRAS and TP53. The allele frequency of most variants increased in enteroid culture, suggesting that cells lacking these mutations failed to propagate (including stromal/immune cells), or cells carrying these mutations expanded at a faster rate. Ten mutations not present in the original tumor were acquired over time in enteroid culture. These mutations included a missense variant of TRPS1, a putative prognosticator of colon cancer. The mutations acquired in enteroid culture may reflect genetic instability in the source neoplastic tissue, or the emergence of subpopulations that were below level of detection in the source tissue. Three mutations were present in the tumor and lost in enteroid culture; this presumably reflects the loss of a subset of cells expressing these variants. Conclusion Changes in allele frequencies suggest that neoplasms are heterogeneous, with shifting cell populations that are differentially affected by culture conditions. This heterogeneity can be further interrogated by comparing allele frequencies in the original tumor with those in enteroids established in reduced or enriched media. This platform can provide further understanding of genetic determinants that underlie the risk for colorectal cancer, as well as strategic insights into the enteroid model as a sophisticated system for the study of tumor biology. Citation Format: Michael K. Dame, Shannon D. McClintock, Durga Attili, Becky Simon, Kelly Copley, Stacy Finkbeiner, Christopher Altheim, Jason Spence, Henry Appelman, D Kim Turgeon, Linda C. Samuelson, Dean E. Brenner, James Varani. Establishment and genomic characterization of enteroid cultures from human colonic adenomas and adenocarcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 322. doi:10.1158/1538-7445.AM2015-322

Abstract 3711: Human colon mucosal crypts in culture: a model to assess colon chemoprevention.

Introduction: In order to advance a primary cell culture model of human colonic carcinogenesis we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomatous polyps. Methods: Fresh biopsy samples were transported in culture medium containing 1.5mM Ca 2+ and antimicrobials. The mucosa/submucosa was surgically separated from the muscularis propria and incubated in 10mM dithiothreitol followed by 8mM ethylenediaminetetraacetic acid. Crypts were agitated free, washed with slow spins, and immediately embedded in a 3-dimensional environment at a density of 50 crypts/50μl Matrigel. After 3 hours, media was replaced to remove non-viable cellular products. Serum-free medium, containing low Ca 2+ (0.15mM), was used to maintain crypt cultures. Cell proliferation and differentiation were assessed with markers Ki67 and E-cadherin by confocal microscopy. The fluorescence signal was quantified for the bottom 1/3 of the crypts (35 cells from base on one crypt edge) using morphometric imaging software. In parallel studies, abnormal crypt-like structures were isolated from adenomatous polyps/adenocarcinoma and maintained in a similar culture system. Results: At day-1 in culture, Ki67 expression was strong in the proliferating cells at the crypt base, with E-cadherin staining increasing towards the crypt apex. By day-3, E-cadherin expression became much more prominent and Ki67 decreased dramatically. This culture system preserved the in vivo phenotype of the crypts and allowed for the colonic epithelial cells to follow the physiological sequence of progression from proliferation to differentiation. Adenomatous tissue in culture at day-1 showed expression for Ki67 and E-cadherin but lacked spacial organization. Conclusion: Intact colonic crypts from normal and abnormal human mucosa can be viably maintained in 3-D culture for 72 hours. This primary human in vitro 3-D model may be used to model efficacy of colonic cancer chemopreventives. Citation Format: Michael K. Dame, Yan Jiang, Danielle Kim Turgeon, Henry Appelman, Muhammad N. Aslam, Kelly Copley, Durga Attili, Dean E. Brenner, James Varani. Human colon mucosal crypts in culture: a model to assess colon chemoprevention. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3711. doi:10.1158/1538-7445.AM2013-3711