Stem Cell Organoids in Primary Cultures of Human Non-Malignant and Malignant Colon

Aims: A sub-population of cells named cancer stem cells (CSCs) that initiate and promote tumour growth have been demonstrated to exist in several malignancies including colon carcinoma. The objective of our pilot study was to isolate CD133+CD26+CD44+ CSCs from patient colon tumours, culture spheres or organoids and observe their proliferation in primary cultures. Parallel cultures of non-cancer controls from colon normal lining and nonadenomatous polyps were set up. Methods: Magnetic activated cell sorting was used to isolate CD133+CD26+CD44+ cell populations followed by primary cell culturing under stem cell culture conditions. Number, cells/organoid and daughter generations of organoids were calculated using phase contrast microscope. Trypan blue exclusion method was used to test the viability of the cells. Results: Both colon tumour and colon non-adenomatous polyp formed floating organoids in suspension; however non-adenomatous polyp cultures did not show self-renewal properties for more than 1 passage. Normal colon singlecell suspension did not create organoids. Metastatic colon tumours rapidly produce cancer cell organoids in less than 24 hours in larger numbers compared to non-metastatic colon tumours (1-3 weeks). Metastatic colon tumour organoids have the ability for proliferation for upto five daughter generations in primary culture compared to three generations for those grown from non-metastatic tumours. Conclusions: This in vitro CSC organoid model will help study colon cancer biology, in particular providing a valuable source of primary cell-derived tissue for studying personalized molecular profiling using ‘omics strategies to direct therapeutic intervention.


Introduction
Colorectal cancer (CRC) is the 3 rd most common cancer type worldwide and the 4 th most common cause of cancer-related mortality with almost 694,000 people dying annually [1]. According to GloboCan 2012 estimates, colorectal malignancies constituted 10.0% and 9.2% of all cancer cases in men and women, respectively. More than half of these cases occurred in developed countries where mortality was substantially lower than the incidence rate. However, more CRC related deaths (52% in 2012) occurred in countries scoring low on the development index, reflecting relative poor survival in these regions [2]. It has been estimated that there were 95,270 new cases of colon cancer diagnosed in USA and 90% to 92% of patients were treated surgically [3][4][5]. Colorectal cancer incidence and death rates declined by approximately 3% per year in both men and women from 2003-2012, with momentum gained in the most recent years likely driven by the increased uptake of colonoscopy screening [5]. In Asia, the incidence of colorectal carcinoma is similar but lower than found in the developed countries. The risk of developing the disease is alike in both sexes at present, however a 41% rise in incidence was noted in the males between 1995-1999 [6]. In a single-center retrospective study of adult patients with colon cancer diagnosed through 2000-2003 (93 patients; M: F=1.58: 1, mean age of diagnosis 54 years), 49.5% of the patients had right sided (mortality rate 51.6%), 10.8% had transverse colon (mortality rate 37.5%), 7.5% had descending colon (mortality rate 66.7%) and 32.2% had sigmoid colon (mortality rate 40.9%) cancers [7]. At Shaukat Khanum Memorial Cancer Hospital and Research Centre (SKMCH&RC, Pakistan), CRCs were among the top five malignancies (3530 cases, 4.91%) diagnosed amongst all age-groups, both sexes combined registered from 1994 to 2015.
Poor survival rate is in part attributed to limited understanding of the characteristics of resistant cancer cells which are now termed cancer stem cells (CSCs) or tumour initiating cells (TICs). The evidence for the existence of CSCs has been reported in blood borne cancer (CD34 + CD38 -) [8], in solid tumour including breast (CD44 + CD24 -) [9,10], brain (CD133 + ) [11], prostate (CD44 + ) [12] and pancreatic (CD44 + CD24 + ESA + ) [13]. The first report on colon cancer stem cells showed that tumourigenic cells were contained within in the rare undifferentiated population that express CD133 [14]. A surface marker profile for isolated CRC stem cells was reported to be epithelial cell adhesion molecule (EpCAM) high /CD44 + /CD166 + [15]. Identification of CD133 + CD26 + CD44 + colorectal CSC is a relatively recent report. A study of 43 patient tumours (27 without liver metastasis, 5 with synchronous liver metastasis on presentation and 15 metachronous liver metastases) analyzed the expression of these markers in dissociated tumour cells by three-color flow cytometry [16]. The most important finding of this study is the ability of CD26 + cells to cause liver metastasis when injected into the cecum of mice irrespective of CD133 or CD44 expression. However, co-expression of CD133 and CD44 did increase the tumourigenic capacity of CD26 + cells. Transient transfection of CD26 siRNA decreased migratory and invasive capacities of the CD26 + CSCs suggesting that CD26 plays a functional role in the invasive capacity of CD26 + CSCs.
CD44 is overexpressed in colon cancers, which often have hyperactivation of the WNT signaling pathway. Deletion of the WNT target, CD44, in Apc(Min/+) mice attenuated intestinal tumourigenesis [17]. CD44, a class I transmembrane glycoprotein that binds to hyaluronan, with various roles from adhesion to signaling [18], is known as a marker of tumour aggressiveness playing a functional role in metastasis [19] and is also a promising marker of CSCs [20]. However, CD44 expression is not limited to CSCs, as many non-CSCs can also express CD44. Hence, combination of CD44 with other markers is used to identify potential CSCs [21]. CD44 expression has been associated with poor patient survival in multiple cancer types, including colon cancers [18]. In intestinal stem cells and in transit-amplifying cells distinct CD44 isoforms contribute to tumour formation [22].
CD133 is a five-transmembrane glycoprotein and identified as a marker of neural CSCs and normal primitive cells of the hematopoietic, epithelial and endothelial lineages [23][24][25]. Interestingly, CD133 + cells from colon tumours have stem-like properties and grow exponentially in vitro as undifferentiated tumour organoids. In colon cancer, they account for approximately 2.5% of the tumour cells [14] and can initiate tumour growth in immune-deficient mice [26]. Recent findings indicated CD133 + cells comprised of both stem-like and differentiated progenitor cells suggesting the CD133 + sub-population (with a combination marker) had multi-functional roles. Co-expression of epithelial cell adhesion molecule (EpCam) and CD44 + in CD133 + fraction further differentiates a sub-group which possess tumourigenic properties [15].
CD26 or Dipeptidyl-peptidase IV (DPPIV) is a serine peptidase involved in cell differentiation, adhesion, immune modulation and apoptosis, all functions which control neoplastic transformation, and its' up-regulation was observed in colorectal tumours [27]. An antimigratory and invasive effect was found with ATP-competitive pan Raf265 inhibitor treatment in combination with 5FU by targeting CD26 + CRC cells [28].
In the present study, we used primary colon tissues to isolate CSC and characterize cultured organoids.

Materials and Methods
All chemicals were obtained from Sigma-Aldrich unless stated otherwise.

Patient specimen collection
A total of 22 colon specimen were obtained (following consent) from patients, undergoing screening colonoscopy and/or invasive surgery from 2015-2016, at SKMCH&RC, Pakistan. Ethical approval of the study was obtained from the Internal Review Board on Human Research at SKMCH&RC. Specimens of normal colonic mucosa and colon nonadenomatous polyp biopsies (2 mm 3 to 5 mm 3 ), to be used as noncancer controls, were collected from non-cancer patients undergoing screening colonoscopy. Specimens were collected within 30 minutes of resection and placed in RMPI medium with 5x antibiotic (500 µg/mL streptomycin, 500 Units penicillin and 1.25 µg/mL amphotericin-B) immediately. A part of the tissue was fixed in formalin for embedding into paraffin wax (for histo-pathological diagnosis) and the remaining tissue was either processed for isolation of cells or stored at -80°C in 1x phosphate-buffered saline (PBS). Colon tumour tissues were obtained during laparoscopy or open surgery performed for tumour resection. These specimens were collected and processed as described above. The manifestation of normal, hyperplastic or neoplastic cells in the attained specimen was confirmed by two independent histo-pathologists. The clinic-pathological features of patients are summarized in Table 1.

Single-cell suspension formation from colon tissues
Mucus containing samples were incubated in sputolysin reagent (100 mM PBS (pH 7.0), 6.5 mM Dithiothreitol) for 15 minutes at room temperature and rinsed with Hank's balanced salt solution (HBSS). A single-cell suspension of each biopsy was generated using the method of Pang et al. [16]. Briefly, fresh specimens were washed with 1xPBS, minced on ice and transferred to 15 mL centrifuge tube containing Dulbecco's Modified eagles Medium (DMEM), combination of Hyaluronidase (Calbiochem, Heidelberg, USA) and Collagenase IV (Invitrogen, Paisley, UK) for enzymatic disaggregation. Complete digestion was ensured by stirring the mixture every 15 minutes and incubation at 37°C for up to 3 hours. The cellular debris in the resulting single-cell suspension was filtered using 40 µm nylon mesh and washed twice with HBSS/2% Hank's buffered salt solution in heat inactivated calf serum (HICS). Histopaque-1077 centrifugation as per the manufacturer's instructions was used to remove red blood cells.

Magnetic bead affinity cell sorting (MACS) of CD133 + CD26 + CD44 +
From the resulting single-cell suspension, CD133 + CD26 + CD44 + were isolated using a Miltenyi Biotech kit as per the manufacturer's instructions. Briefly, single-cell suspensions were incubated with a monoclonal CD133-APC (Miltenyi Biotech, Bergisch Gladbach, Germany) antibody, labeled with MicroBeads (Miltenyi Biotech) for 30 min at 4°C. Anti-APC Multisort Kit (Miltenyi Biotech) was used to obtain the CD133 + cells followed by cleavage from the MicroBeads using release reagent from the kit. CD133 + cells were incubated with CD26-PE (Miltenyi Biotech) antibody and Anti-PE Multisort Kit (Miltenyi Biotech) was used to obtain CD133 + CD26 + cells. The microbeads were cleaved from the CD133 + CD26 + cells by using the release reagent in the Multisort kit. The CD133 + CD26 + cells were incubated with the CD44 Microbeads (Miltenyi Biotech,) to obtain CD133 + CD26 + CD44 + cells. The filtrate was collected containing CD44 -CD26 -CD133cells. Primary culture of CD133 + CD26 + CD44 + cell organoids Separate cultures for each type of fresh specimen (colon normal lining, colon non-adenomatous polyp and colon tumour) were set up.

Self-renewal assay
The organoids, collected by centrifugation and digested to singlecell suspension using Dispase II (20 U/mL of DMEM), were filtered through 40 μm filter. For daughter organoids formation and colony formation, 250 cells/cm 2 in 10 mL of medium were cultured in nonadherent or adherent 25 cm 2 culture flasks, respectively. The organoids in suspension and adhesive colonies were observed using phase contrast microscope (Labomed TCM 400). Trypan blue exclusion method was used to test the viability of the cells.

Results
Organoid formation in primary cultures of CD133 + CD26 + CD44 + Cells The single-cell suspension from each type of colon normal lining (Figure 1a), colon non-adenomatous polyp (Figure 1d) and colon tumour (Figure 1g and 1j) was incubated in non-adherent stem cell medium to observe their organoid formation patterns in primary cell culture. Purified CD133 + CD26 + CD44 + cells exhibited sphere-forming abilities compared to the CD133 -CD26 -CD44equivalents. The MACS isolated cells (Figure 1b) from non-cancer colon normal lining did not produce characteristic compact round organoids (Figure 1c). The isolated CD133 + CD26 + CD44 + cells (Figure 1e) from colon nonadenomatous polyp showed a slow growth pattern of loosely compact organoids (Figure 1f) which did not survive for more than a month. However, organoids (which mimic tumor in a flask) were generated at much faster rate within 1 day from CD133 + CD26 + CD44 + cells isolated from non-metastatic (Stage II) tumours (Figure 1i) organoids, whereas those isolated from advanced disease metastatic (Stage IV) tumours ( Figure 1l) exhibited highest growth rate and cell numbers in less than 24 hours. After 24 hours of culture, floating undifferentiated CD44 -CD26 -CD133cells start to adhere to the flask (both collagen coated and non-coated) and attain the shape similar to that of colon epithelial cells.

Self-renewal of cancer cell organoids
The ability for self-renewal and adoption of a spherical morphology, attributed to the presence of CSCs, were tested by collecting the CD133 + CD26 + CD44 + cell organoids and culturing them after dissociation into single-cells. The secondary organoids were formed within 1 week from colon non-adenomatous tissue but did not regenerate secondary organoids on 2 nd passage. A progressive loss of regenerative ability by the third passage was observed with primary cells from non-metastatic tumours and by fifth passage for those from metastatic cancers (Table 2).

Discussion
The aim of our pilot study was to isolate CD133 + CD26 + CD44 + CSCs from patient colon tumours, culture spheres or organoids and observe their proliferation in primary cultures. We setup primary cultures from human colon tumour to grow cancer cell organoids, isolated CD133 + CD26 + CD44 + cells by MACS followed by culturing to produce cell organoids. Normal tissues were processed as the tumor tissues using same method of isolation. Under the same culture conditions, the non-cancer cells did not produce organoids while those from nonmetastatic and metastatic tumours produced compact, round and multi-cellular organoids (Figure 1) confirming that only tumour cells have self-renewal abilities. The non-cancer tissue produced single-cells which died after few weeks of culture. The organoids from different tissue sources, survived over different time periods (1-30 days), which could be attributed to the different type of cells with specific set of mutated genes in various tissues (Table 2).
Comprehensive comparisons of gene pool and proteome of these organoids from various stages of cancer can be helpful in monitoring CRC progression and metastasis. These genetic and proteomic differences between non-adenomatous polyps and normal colon lining, which did not translate into identifiable histologic features, could be the reason why non-adenomatous polyps demonstrated a loosely compact sphere formation pattern (Figure 1f) absent from normal colon lining cultures. This organoid model may provide the means for in-depth 'omics' analyses to understand the dynamics of the cancer.

Conclusion
In summary, we successfully isolated CD133 + CD26 + CD44 + cells from human colon tissues by magnetic beads and generated colon cancer cell organoids in primary cultures in supplemented medium. The potential applications for colon organoids (potentially harboring CSCs) characterization include developing methods for CSC detection, CSC resistance to cancer therapies, identifying new potential therapeutic targets and study mechanism of suppression of cancer metastasis.  None Cells plated at a density of 250/cm 2 in 10 mL of conditioned media in Ultra-Low attachment culture flasks Triplicate analyses were performed from each sample type NA: Not applicable