Aberrant activation of the CD45-Wnt signaling axis promotes stemness and therapy resistance in colorectal cancer cells

Rationale: Chemoradiation (CRT) is commonly used as an adjuvant or neoadjuvant treatment for colorectal cancer (CRC) patients. However, resistant cells manage to survive and propagate after CRT, increasing the risk of recurrence. Thus, better understanding the mechanism of resistant cancer cells is required to achieve better clinical outcomes. Methods: Here, we explored gene expression profiling of CRC patient tumors to identify therapy resistance genes and discovered that protein tyrosine phosphatase receptor type C (PTPRC), which encodes CD45, was increased in remnant tumor tissues after CRT and correlated with metastasis. Through multiple validations using patient tumors and CRC cell lines, we found for the first time the increase of CD45 expression in CRC (EpCAM+) epithelial cells surviving after CRT. Thus, we investigated the biological role and downstream events of CD45 were explored in human CRC cells and CRC mouse models. Results: Increased CD45 expression in cancer cells in pretreated primary tumors accounts for poor regression and recurrence-free survival in CRT-treated patients. High CD45 expression promotes CRC cell survival upon 5-fluorouracil or radiation treatment, while CD45 depletion sensitizes CRC cells to CRT. Intriguingly, CD45 is preferentially expressed in cancer stem-like cells (CSCs), as determined by spheroid culture and the expression of CSC markers, and is required for the distinct functions of CSCs, such as cancer initiation, repopulation, and metastasis. Mechanistically, CD45 phosphatase activity promotes Wnt transcriptional activity by stabilizing the β-catenin protein, which collectively enhances stemness and the therapy-resistant phenotype. Conclusions: Our results highlight a novel function of CD45 as a mediator of CRT resistance and provide a potential therapy strategy for CRC therapy.

CD45 protein levels were determined in the same cell lysate samples used in Figure 1G by performing a Western blot analysis with different anti-CD45 antibodies (#ab8216). β-Actin served as a loading control. (I) Cells were treated with 5-fluorouracil (5-FU, 1 μM) and radiation (4 Gy). After 48 h, the surviving cells were subjected to Western blot analysis to determine CD45 protein levels (#10558, Abcam). γH2AX (#11175, Abcam) was used as a marker for DNA damage induced by 5-FU or radiation. Statistical analyses comparing two groups were performed using Student's t-test. *** indicates p-values <0.001.  (A) Patient-derived primary CRC cells were separated into two groups according to the CD45 expression level (CD45 high and CD45 low ). The IC50 values of 5-FU were determined using an MTT assay (n = 5/group) and calculated using GraphPad Prism 5 software (GraphPad Software, Inc., CA, USA). (B) Radiation sensitivity was measured using traditional methods, in which the survival potential of irradiated cells was estimated with clonogenic assays, and the radiation biological parameters and statistical significance were then analyzed using a linear-quadratic model (n = 3/group). The parameters and statistical values were calculated using GraphPad inhibitor were determined as described in (B). (E) PTPRC mRNA levels were determined using RT-qPCR after 5-FU or radiation treatment with or without CHK1 the inhibitor (0.3 μM, n = 3/group). (F) CD45 knockdown experiments were performed with siRNAs targeting the PTPRC gene (siPTPRC). At 24 h after siRNA transfection, total RNA was extracted and subjected to RT-qPCR. Significant reductions in PTPRC mRNA levels were determined using one-way ANOVA with Dunnett's multiple comparison tests (GraphPad Prism 5, n = 3/group). (G) Decreases in CD45 protein levels were confirmed using Western blotting. (H) FACS analysis validated the reductions in CD45 protein levels after CD45 knockdown (n = 3/group). Statistical significance was determined as described in (F). (I) The most potent siPTPRC sequence (#3) was cloned into the pLKO-puro lentiviral vector to generate an shRNA plasmid (shPTPRC). The shPTPRC vector was transfected into the HCT116 cell line, and stable cells were generated by antibiotic selection. CD45 knockdown efficiency was confirmed using RT-qPCR and Western blot analyses (n = 3/group). (J) HCT116 and (K) DLD1 cells overexpressing (OE) CD45 were generated via lentiviral vector transfection (LV277909, abmGood, Vancouver, Canada). After antibiotic selection, CD45 overexpression was confirmed using RT-qPCR and Western blot analyses (n = 3/group). (L) Western blot analyses were performed immediately after or 24 h after radiation exposure using lysates from control, CD45-OE, and CD45-depleted HCT116 cells. μM, 48 h), radiation sensitivity was measured as described in (B, n = 3/group). (R-U) The percentage of apoptotic cells at 24 h after 5-FU or radiation treatment was visualized (R and S) by performing Annexin V staining (n = 3/group) or (T and U) by visualizing the level of cleaved PARP with or without NQ-301 treatment (0.5 μM, 24 h). Statistical analyses were performed using one-way ANOVA followed by Dunnett's multiple comparison tests for comparisons to the control group or Student's t-test for comparisons between two groups. ** and *** indicate p<0.01 and p<0.001, respectively. The incidence of tumor-bearing mice was monitored for 28 days after the inoculation of (D) patient-derived CRC cells (P#6441493) or (E) HCT116 cells. The definitive tumor volume was determined by necropsy on day 28. Statistical significance of differences between two groups was determined using Student's t-test. *, ** and *** indicate p<0.05, p<0.01 and p<0.001, respectively. (F) Sphere-forming potentials were compared between CD45 high and CD45 low cells isolated from HCT116 spheres generated from CD45 high cells, as shown in Figure 4H. Cells were seeded at varying cell densities and incubated under sphere culture conditions for 14 days, and then the number and size of spheres were counted (n = 6/group). (G) FACS plots show the increase in the CD45 high population under sphere culture conditions from approximately 6% to 30%. HCT116 spheres were dissociated into single cells and divided into two groups according to the CD45 expression levels (CD45 high and CD45 low cells). These cells were seeded and incubated under sphere culture conditions for 14 days to generate spheres (the first-passage spheres). These first-passage spheres were dissociated into single cells and subjected to FACS to analyze CD45 expression levels and then proceeded to a second-passage sphere formation assay.
The first-passage spheres from the CD45 high population consisted of both CD45 high and CD45 low populations with a ratio similar to that of the initial CD45 high and CD45 low populations (approximately 30%), while the spheres generated from the CD45 low population consisted of only a CD45 low population. Consistently, in the second-passage spheres, the generation of CD45 low cells from a CD45 high population was confirmed with a ratio similar to that of the initial CD45 high and CD45 low cells. (H) Immunohistological staining of CD45 expression levels in HCT116 xenograft tissues generated from CD45 high and CD45 low populations ( Figure 4E). In this experiment, carcinoembryonic antigen (CEA), a CRC marker, was used to identify CRC cells in tissues. EpCAM was used to validate their epithelial origin. (I) In vitro limiting dilution assays were performed to compare the sphere-forming potential between NQ-301-treated (0.5 μM, 14 days) or control (DMSO) CRC cells (n = 12/group). Statistical analyses comparing differences between two groups were performed using Student's t-test. *, ** and *** indicate p-values <0.05, <0.01 and <0.001, respectively.  3/group). In addition, the sensitivity of the cells to radiation was measured using traditional methods; the survival potential of irradiated cells was estimated with clonogenic assays, and the radiation-related biological parameters and statistical significance were then analyzed using a linear-quadratic model (right panel, n = 3/group). Statistically significant differences between the two groups were calculated using GraphPad Prism software version 5. (H) An in vitro limiting dilution assay was performed to compare the sphere-forming potential between β-cateninknockdown and control CRC cells (n = 12/group). (I) A sphere-forming assay was conducted to examine the effect of β-catenin knockdown on CD45-induced stemness. Wild-type or CD45-OE cells were seeded at varying cell densities after siCTRL or siCTNNB1 transfection and incubated under sphere culture conditions for 14 days. Then, the number and size of spheres were counted (n = 6/group). (J) A sphere-forming assay was conducted to confirm the involvement of β-catenin in CD45-induced stemness using ICG001, an inhibitor of β-catenin-mediated transcription. EVtransfected or CD45-OE cells were seeded at varying cell densities and incubated under sphere culture conditions with or without ICG001 (1 or 10 μM) for 14 days. Then, the number and size of spheres were counted (n = 6/group). Statistical analyses comparing results with the control group were performed using one-way ANOVA followed by Dunnett's multiple comparison tests or Student's t-test for comparisons between two groups. *, ** and *** indicate p-values <0.05, <0.01 and <0.001, respectively.
*Bold sequences were used to generate shRNA plasmids.

Chemicals
Radioimmunoprecipitation assay (RIPA) buffer was prepared in our laboratory as

Visualization of scRNA-seq data
The scRNA-seq data from CRC tumors were obtained from the GEO web server

FACS analysis
FACS analysis was performed using a BD Accuri TM flow cytometer (BD Biosciences).
FACS data were analyzed using FlowJo software (TreeStar, San Carlos, CA, USA) as described in our previous report [1]. Cells were stained with specific antibodies according to the manufacturers' instructions. After a 1 h incubation at 4 °C, cells were washed with phosphatebuffered saline (PBS) and analyzed using a BD FACSCalibur system (BD Biosciences). The detailed antibody list is provided in Table S3.

Isolation of primary cells from normal intestines of wild-type mice and from adenomatous polyps of APC Min/+ mice
Normal intestines and adenomatous intestinal polyps were surgically removed from wildtype and APC Min/+ mice, respectively. We detached intestinal epithelial cells from the basement membrane using a previously reported EDTA/DTT protocol with slight modifications [2].

Comparison of relative sensitivity to 5-FU or radiation
Relative sensitivities to 5-FU were compared by determining the half-maximal inhibitory concentration (IC50) values based on reductions in cell viability. Briefly, cells were seeded in 96-well plates (5,000 cells/well) and incubated for 24 hours for attachment. Then, the cells were treated with 5-FU at various concentrations and incubated for 48 hours. Cell viability was measured by staining with thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA), and the absorbance was measured using a microplate spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT, USA). In addition, the sensitivity of the cells to radiation was measured using traditional methods [3]. Briefly, the survival potential of irradiated cells was estimated with clonogenic assays, and the radiation-related biological parameters and statistical significance were then analyzed using a linear-quadratic model (GraphPad Prism version 5, GraphPad Software, San Diego, CA, USA). Cell proliferation was measured using a fluorescence-based BrdU labeling and detection kit (Thermo Fisher Scientific) based on a 2-hour incorporation of BrdU.

Retrospective study population of rectal cancer for determining the correlation between marker expression and therapeutic response to preoperative chemoradiotherapy.
The clinical validation study set consisted of 44 pretreatment paraffin-embedded tissue samples obtained from patients with locally advanced distal rectal cancer (cT3-T4, N+) who had been treated at Chungnam National University Hospital. Clinicopathological characteristics are summarized in Supplementary Figure S2B. The cases had been previously reported [4]. Among the previously reported 93 cases, 53 cases with sufficient amount of cancer tissue were arranged in one tissue microarray block containing one representative tissue core of 2mm in diameter from each case. Out of the 53 cases, 9 cases were excluded from the analysis, since 5 cases were not available for tissue within the core, 2 cases showed no more cancer gland within the core, and the other two were poor quality of tissue with artifact. All cases were histologically proven low-grade (well to moderately differentiated) adenocarcinomas and the patients had received preoperative chemoradiotherapy (CRT) consisting of 50.4 Gy of pelvic irradiation in 28 fractions, combined with 2 cycles of 5-fluorouracil (5-FU) (400 mg/m 2 per day) or capecitabine (1650 mg/m 2 per day) and leucovorin (20 mg/m 2 per day), followed by curative surgery average 6 weeks after completion of CRT. The surgically resected cases were pathologically diagnosed according to WHO classification [5], and were classified according to AJCC TNM system [6].

Gene expression modification by knockdown or overexpression
Cells were transfected with siRNAs targeting specific genes or a nonspecific negative control siRNA (Bioneer, Daejeon, Republic of Korea) in media (serum-, phenol-, antibiotic-free) with Lipofectamine TM 2000 (Invitrogen) according to the manufacturer's instructions.
Knockdown efficiency was confirmed by measuring mRNA expression using reverse transcription PCR and RT-qPCR. The sequences of the siRNAs are listed in

Immunofluorescence staining of cells
Proteins were visualized using the specific antibodies described in Table S3.

RNA isolation, reverse transcription PCR, and RT-qPCR
Total RNA was isolated from mouse tissues or cells using TRIzol reagent (Invitrogen).  Table S5.  Table S3.

Apoptosis assay (Annexin V+)
The rate of cell apoptosis was quantitatively analyzed by performing apoptosis assays using an Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit I (BD Biosciences). Cell suspensions (1 x 10 6 /mL) were prepared by washing cells twice with cold PBS. Then, 100 μL of the suspension were transferred to a tube to which 5 μL of FITC, Annexin V, and propidium iodide (PI) were added. The mixture was incubated at room temperature for 15 min in the dark after gentle vortexing. After incubation, 400 μL of 1X binding buffer were added before analysis using flow cytometry.  Table S3.

Luciferase reporter assays
A TOP-FOP luciferase assay was performed as described in our previous report to

IP
IP was performed specifically for β-catenin to detect its ubiquitination. Cells were harvested by adding complete RIPA buffer to the plate. The supernatant of the sample was considered the cell lysate. The concentration of the cell lysate was adjusted to 1 μg/μL and then reacted with a β-catenin antibody (Cell Signaling) or normal rabbit IgG (Cell Signaling). Protein A agarose beads were added to each β-catenin antibody, normal rabbit IgG, and control sample (input) and bound by rotating at 4 °C. After the bead binding step, lysates bound to antibodies were centrifuged, and their pellets were resuspended and prepared for Western blotting. Mouse anti-rabbit IgG (light-chain specific) was used as the secondary antibody to detect β-catenin.

In vivo limiting dilution assay (LDA)
For the comparison of the tumor-initiating potential between CD45 high and CD45 low cells, patient-derived primary CRC cells or HCT116 cells were sorted into two groups according to the CD45 expression level by FACS (CD45 high and CD45 low ), and the cells were then subcutaneously inoculated into NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ, #005557, Jackson Laboratory, Bar Harbor, ME, USA) at various cell densities. After 28 days of observation, the frequency of tumor formation was monitored for 28 days and definitely determined by necropsy (n = 8 animals/group).
For the comparison of tumor-initiating potential between PTPRC knockdown and control HCT116 cells, cells were subcutaneously inoculated into NSG mice. On day 35, the mice were sacrificed, and the primary tumors were removed. Single tumor cells were isolated from the primary tumors by depleting mouse stromal cells with a mouse cell depletion kit (Miltenyl Biotec, Bergisch Gladbach, Germany), and then CRC cells were subjected to a limiting dilution assay to test their tumor-repopulating capability. The incidence of tumors in mice was monitored for 16 weeks and determined by definitive necropsy. LDA graphs were generated and statistical values were calculated using online software provided by Walter+Eliza Hall Bioinformatcis (http://bioinf.wehi.edu.au/software/elda/) as described in a previous report [8].

In vitro LDA and sphere-forming assays
For the in vitro sphere-forming assay, cells were seeded at varying cell densities and incubated under sphere culture conditions (poly-HEMA-coated 96-well plates; poly 2hydroxyethyl methacrylate, Sigma Cat # P3932) for 14 days, and then the number of wells without spheres was counted (n = 12/group). The generation of LDA graphs and statistical calculations were performed using online software provided by Walter+Eliza Hall Bioinformatcis (http://bioinf.wehi.edu.au/software/elda/) as described in a previous report [8].
For the sphere formation assay, cells were seeded at varying cell densities and incubated under sphere culture conditions (poly-HEMA-coated 6-well plates) for 14 days, and then the number and size of spheres were counted (n = 6/group).

Splenic injection mouse model
A splenic injection experiment was performed to estimate the step governing metastasis and distant organ colonization [9]. In this model, shCTRL-or shPTPRC-transfected HCT116-luc cells (1X10 6 cells/mouse) were inoculated into the spleen followed by splenectomy, and the surviving cells that grew in distant organs then contributed to the formation of liver metastases.
We routinely monitored liver metastasis weekly by visualizing luciferase activity for 28 days (n = 9 for shCTRL, n = 8 for shPTPRC). After sacrifice, the livers were removed to determine liver metastasis.

APC Min/+ mouse polyp-derived organoid culture
Single cells were isolated from the intestinal polyps of 20-week-old APC Min/+ mice and Then, the transfected cells were cultured and monitored as described above. To test the effect of CD45 pharmacological inhibition on organoid growth, NQ-301 (0.5 μM) was treated on the 1 st day after seeding, and measured the organoid viability on the 7 th day of organoid culture as described above.

Migration and invasion assay
The

Protein-protein interaction docking simulation
The possible molecular interaction between the CD45 (PDB code: 1YGR) and β-catenin (PDB