Colorectal cancer (CRC) is the third most common type of newly diagnosed cancer and the second leading cause of cancer-related deaths worldwide.
1 Patients with early-stage CRC are primarily treated surgically, whereas those with advanced CRC require additional perioperative radiation therapy and chemotherapy.
2 Although radiation therapy and chemotherapy may be curative in a number of cancer types, success is limited by the development of resistance. Cancer stem cells (CSCs) are one cause of this problem. CSCs were first noticed in acute myeloid leukemia.
3 The de-differentiation and transformation of normal cancer cells into stem cell-like cells may be the mechanism that induces the development of CSCs.
4,5
Cells within a tumor have diverse phenotypic systems, and therapeutic resistance is implicated in this diversity.
6,7 CSCs contribute to this diversity. Conventional chemotherapy targets non-CSCs in the tumor and fails to eliminate CSCs, resulting in limited efficacy.
8‐10 This is evidenced by CSCs being more resistant to conventional therapies than non-CSCs.
11,12 Even treatments that completely eliminate non-CSCs may be able to repopulate tumors if only CSCs remain.
7,13
CD44 has been proposed as an important cancer stem cell marker in several cancers.
14,15 CD44 is a cell surface glycoprotein that plays roles in the adhesion of the cytoskeleton to the extracellular matrix, cell–cell interactions, and cell migration.
15-17 CD44 knockdown has been reported to prevent tumor formation and clonogenesis.
18 The ability of CD44+ve/CD24+ve cells to differentiate into the enterocyte, enteroendocrine, and goblet cell lineages in vitro also has been established.
19 CD44 overexpression has been linked to high cancer aggressiveness and resistance.
20
CSCs, with their unique surface markers, have unique properties that protect them against cytotoxic drugs. Therefore, investigations into CSCs may help identify the population that is more resistant to treatments. Identification of resistant CSCs after chemotherapy is very helpful for the treatment of refractory tumors, and the investigation of the surface markers is as well. This study evaluated persisting CSCs (even after stimulation with chemotherapeutic agents) that cause tumor recurrence.
Materials and Methods
CRC Cell Line Culture
Human CRC cell lines (DLD-1, HCT116, HT29, RKO, and SW480) were a gift from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD). The cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA), 1% GlutaMAX-I (Thermo Fisher Scientific Inc.), and 1% penicillin/streptomycin/amphotericin B (Wako Pure Chemical Industries Ltd., Osaka, Japan). The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
Establishment and Culture of Human Organoids
CRC tissue was cut into small pieces, dissociated using 1 mg/mL collagenase (C6885; Sigma-Aldrich, St. Louis, MO) in DMEM, and shaken in a bioshaker BR-13FP (Taitec Co., Saitama, Japan) at 6 ×
g for 15 min at 37 °C. Dissociated tissues were filtered through a custom-made filter (Sansho Co., Tokyo, Japan), centrifuged at 400 ×
g for 5 min at room temperature (RT, 20-25 °C), and the collected cell pellets were resuspended in a culture medium (modified stem cell culture medium
21). Suspended human organoids (iCC603, iCC821, and iCC724) were seeded onto plates coated with Matrigel (Corning Inc., Corning, NY). The medium was changed every 2–3 days. After the cells had spread to more than 50% of the plate, they were passaged with Accutase (Nacalai Tesque, Kyoto, Japan) for approximately 5 min. Cells were collected, resuspended in the culture medium, and seeded onto Matrigel-coated plates. Obtaining the medical records and clinical samples, written, informed consent was obtained from all participants following the ethics guidelines of the Osaka International Cancer Institute.
Flow Cytometry
The expression of surface proteins within the collected cells was determined by using flow cytometry (FC). Cells were dissociated with Accutase (Nacalai Tesque) and stained with CD24 (1555427; BD Biosciences), CD44 (103012; BioLegend, 338820; BioLegend), CD44v5 (L MCA1729; Bio-Rad), CD44v6 (MCA1730; Bio-Rad), CD44v7 (MCA1731; Bio-Rad), CD44v9 (LKG-M003; Cosmo Bio), CD133 (372808; BioLegend), and 7-AAD (372808; BD Biosciences). The relative fluorescence intensities were measured by using an SH800 cell sorter (Sony Corporation, Tokyo, Japan). A two-dimensionality reduction step was performed using t-distributed stochastic neighbor embedding (t-SNE) to visualize high-dimensional cell surface marker expression data in a low-dimensional space. Data were analyzed by using the FlowJo software, Version 10.2 (FlowJo).
Time Course Evaluation
For the withdrawal period, we divided human organoids into two groups: one in which chemotherapeutic agents were administered for 3 days, and then, CD44 marker expression was analyzed (=Day 0), and the other in which the medium was changed and CD44 expression was analyzed after 1, 2, and 3 days (=Post 1, 2, and 3 days). For the duration of treatment, human organoids were treated with agents for 1 to 5 days to analyze the expression of CD44 markers.
RNA Preparation and Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
Gene expression microarrays were analyzed in CD44 cells. CD44+ve and CD44-ve cells were sorted using an SH800 cell sorter (Sony Corporation, Tokyo, Japan).
Total RNA was prepared by using an RNA Purification Kit (Qiagen GmbH, Hilden, Germany). Reverse transcription was performed using a Transcriptor First Standard cDNA Synthesis Kit (Roche Diagnostics, Tokyo, Japan). qRT-PCR was performed by using the FastStart TaqMan Probe Master (Roche Diagnostics), the Universal Probe Library platform (Roche Diagnostics), and the CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) for cDNA amplification of target genes. The primers and Universal Probe Library probes used in this study are listed in Supplementary Table S1.
Proliferation Assay
Immediately after FC, 1 × 105 cells of CD44+ve and CD44-ve human organoids were seeded into 12-well plates. Proliferation in the same well was evaluated over time by live-cell imaging using the IncuCyte S3 Live-Cell Analysis System (Sartorius, USA) on adhesion cell fluence.
Drug Sensitivity Assay
Cell lines (2 × 103 cells/well) and organoids (5 × 103 cells/well) were seeded and cultured in 96-well plates or 6-well plates. When cells were 60–70% confluent, they were treated with 5-FU (0.3–150 μg/mL for cell lines and 0.003–300 μg/mL for primary culture cells). After 3 days, cell viability was measured using the CCK-8 assay (Dojindo Molecular Technologies, Inc.).
Xenograft Model for Histological Examination of Primary Cultured Cells
Histological examination of parent and CD44-ve cells after chemotherapeutic agents were performed using a xenograft model. Accutase-dissociated cells (5 × 105 cells) suspended in Matrigel (BD Biosciences, Franklin Lakes, NJ) were subcutaneously transplanted into the dorsal flanks of 7-week-old, male, nonobese, diabetes/severe combined immunodeficiency mice (CLEA, Tokyo, Japan). 2D organoids (2DOs) were injected into different mice. The mice were sacrificed 3 weeks after transplantation, or when the tumor diameters were 15 mm, by cervical dislocation under anesthesia. The mice were weighed weekly, and no mice had reduced body weight. Xenograft tumors were fixed in formalin, processed through a series of graded ethanol concentrations, embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin. After deparaffinization and blocking, sections of the CRC specimen were incubated with primary anti-POU5F1 rabbit polyclonal antibody (#2570; Cell Signaling Technology Inc., Beverly, MA) and primary anti-Ki-67 rabbit monoclonal antibody (ab16667; Abcam, Cambridge, UK) at a dilution of 1:200 overnight at 4 °C. Vectastain Universal Elite (Vector Laboratories, Burlingame, CA) was used to detect the signal. Diaminobenzidine was used for color modification. All sections were counterstained with hematoxylin. The Osaka International Cancer Institute Review Board and Animal Research Committee approved this study.
Establishment of DsRed-transfected Cells
The vector pLV[Exp]-Neo-CMV>DsRed_Express2 (Vector Builder, VB900088-2435mhv) was transfected into 2DOs using the Lentiviral High-Titer Packaging Mix with pLVSIN (Takara Bio Inc., Shiga, Japan), according to the procedure described in our previous report.
22 Subsequently, the DsRed-positive cells were sorted by using an SH800S cell sorter (Sony Corporation, Tokyo, Japan).
Establishment of POU5F1-EGFP-Casp9 Cells
PL-SIN-Oct4-EGFP, which expresses EGFP under the control of the POU5F1(Oct4) promoter, was a gift from James Ellis (Addgene plasmid # 21319).
23 In addition, by pMSCV-F-del Casp9.IRES.GFP, kindly gifted by David Spencer (Addgene plasmid # 15567),
24 we established cells expressing EGFP under the OCT4 (POU5F1) promoter with inducible caspase 9. We digested sequence-encoding caspase 9 with restriction enzymes EcoRI-HF (R3101S; New England Biolabs) and XhoI (R0146S; New England Biolabs, Beverly, MA). The DNA fragment of caspase 9 was extracted from E-Gel CloneWel 0.8% (G6500ST; Thermo Fisher Scientific) using the E-Gel Power Snap Electrophoresis System (Thermo Fisher Scientific).
This fragment was amplified by using CloneAmp HiFi PCR Premix (Z9298N; Takara Bio) with primers (FW_gaattctgcagtcgatcgagggtcaggtgg, RV_ccgcggtaccgtcgacttagtcgagtcgagtcgttagc). Amplification. PL-SIN-Oct4-EGFP was linearized by a restriction enzyme, SalI-HF (R3138S; New England Biolabs). The amplified fragments and linearized vector were used for the cloning reaction by the In-Fusion HD Cloning Kit (Z9648N; Takara Bio). The transformation procedure was performed using Competent High E. Coli DH5α (TYB-DNA903; Toyobo, Osaka, Japan), and the plasmid was extracted using the Qiagen Plasmid Plus Midi Kit (12945; Qiagen). The nucleotide sequence of the vector was confirmed by Sanger sequencing performed by GENEWIZ Japan Corp. (Kawaguchi, Japan). Primer extension sequencing was performed using Applied Biosystems BigDye version 3.1, and the reactions were then run on an Applied Biosystem's 3730xl DNA Analyzer. The constructed vector was transfected into two PDOs (iCC603 and iCC724) by using Lentiviral High Titer Packaging Mix with pLVSIN (Takara Bio). EGFP-positive cells were cloned by single-cell sorting using an SH800 cell sorter (Sony Corporation, Tokyo, Japan). POU5F1 expression was confirmed by PCR, and a decrease in the number of EGFP-positive cells was evaluated by the administration of B/B Homodimerizer (Z5059N; Takara Bio) (dimerizer). The mean provirus copy number was 6.05 (±1.16, n = 6), as measured using the Let-X Provirus Quantitation Kit (Z1239N; Takara Bio).
Single-Cell RNA Sequencing of Human Organoids and Generation of Data Matrix
Single-cell library preparation was performed following the manufacturer’s instructions for the Chromium Next GEM Single Cell 3′ Reagent Kits (v3.1; 10x Genomics, Pleasanton, CA), and the libraries were sequenced on a HiSeq X sequencer (Illumina, San Diego, CA). Cell Ranger pipeline (version 6.1.2) was applied to generate the data matrix. Raw reads were aligned to the human reference genome (GRCh 38) by using STAR aligner. Seurat (version 4.1.0) was used for quality control and downstream analysis. Poor quality cells were filtered out using the following parameter: nFeature_RNA 1000 – 7000 and percent.mt < 15, and finally, 3,654 cells that passed QC were finally used for further analysis. Uniform manifold approximation and projection (UMAP) visualization was used for dimensionality reduction analysis with the following parameters: resolution 0.5 and perplexity 40. Marker genes discriminating the different clusters were identified by using the FindAllMarkers function. To calculate RNA velocity, the velocyto R package (v0.6) was applied.
Statistical Analysis
Continuous variables were expressed as means ± standard deviations or standard errors of the means. Student’s t tests were used to analyze the differences between two independent groups. All statistical analyses were performed by using JMP (SAS Institute Inc., Cary, NC). P values < 0.05 were considered statistically significant.
Discussion
Unlike cell lines, clinical tissues are heterogeneous populations and can be evaluated in primary cultured cells as a model of tumor heterogeneity (i.e., diversity).
25‐27 Upon chemotherapeutic agent stimulation, CD44-ve cells appeared in organoids but not in the cell lines. These results that newly emerged CD44-ve did not appear in the cell lines suggest that these newly emerged cells depend on the original “diverse” cell population. These unique, emerged cells also had CD44 but not stem cell markers, such as CD24 and CD133. We examined whether the presence of these cells led to resistance to chemotherapeutic agents. CD44 is expressed in many cells and is involved in cell adhesion and migration and in regulating lymphocyte kinetics, such as lymphocyte rolling in immune responses.
15,16,28 CD44 is known as overexpressed in CRC and has been recognized as a molecular marker of CSCs. CD44 also has several variants that are considered markers of CSCs.
29,30 Traditionally, CSCs present in the tumor are positive for CD44, which is considered a CRC stem cell marker, and these cells are resistant to chemotherapeutic agents.
31 It is believed that these stem cells survive and rebuild their original population after chemotherapeutic agent stimulation, exacerbating resistance to chemotherapeutic agents.
32 CD44+ve cells have been reported to be chemotherapy-resistant as CSCs, but there are no reports on the unique CD44-ve cells that appear transiently after this chemotherapy. Different organoids have different concentrations of CD44-ve appearing. We consider that this is the cause of resistance, because this population appears at higher concentrations and not at lower doses of chemotherapeutic agents. In this study, we hypothesized that CD44-ve cells exist in tumors in addition to conventional CSCs and that these cells, which emerge when the chemotherapeutic agents’ concentration and overall tumor stress increase, are involved in tumor growth and resistance after chemotherapeutic agents. We hypothesized that these cells generate stem cells independently, which would support CSCs when they survive drug administration.
CD44-ve cells reestablish and form a similar population to the original (parental) population, and perhaps, the CD44-ve population contains cells that induce CSCs (i.e., cells that are the source of CSCs). After chemotherapeutic agent stimulation, CD44-ve cells may be derived from CD44+ve cells within the parental line. These results indicate that the emerged CD44-ve cells were derived from CD44+ve cells before chemotherapy, consistent with previous reports.
14,15 It suggests that the emerged population is the “true” CSCs causing drug resistance. After chemotherapeutic agent stimulation, CD44-ve cells have a higher proliferative capacity and are more malignant than CD44+ve cells. Single-cell analysis showed a higher percentage of cells with POU5F1 (OCT4) expression within the CD44-ve cell population and high stemness after chemotherapeutic agents, such as 5-FU for POU5F1 in vivo. POU5F1 was highly expressed in transient CD44-ve cells, and suppressing the POU5F1 leads to the treatment for the prevention of the recurrence/relapse after chemotherapeutic agent. POU5F1 is likely involved in chemotherapeutic agent resistance to the stemness.
For the treatment against refractory tumors, such as the recurrence after chemotherapeutic agents, the treatment should target the emerging specific population such as CD44 (or CD44v9) as well as proliferative cancer cells.
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