CD44 Expression Level and Isoform Contributes to Pancreatic Cancer Cell Plasticity, Invasiveness, and Response to Therapy

Published OnlineFirst June 7, 2016; DOI: 10.1158/1078-0432.CCR-15-3115

Biology of Human Tumors Clinical Cancer Research CD44 Expression Level and Isoform Contributes to Pancreatic Cancer Cell Plasticity, Invasiveness, and Response to Therapy Shujie Zhao1, Chen Chen1, Katherine Chang1,2, Anand Karnad1,2, Jaishree Jagirdar3, Addanki P. Kumar4,5, and James W. Freeman1,2,5

Abstract

Purpose: A subpopulation of pancreatic ductal adenocarci- Results: We identiﬁed an invasive cell population that gives rise noma (PDAC) cells is thought to be inherently resistant to to gemcitabine-resistant tumors. These cancer cells express a high chemotherapy or to give rise to tumor cells that become level of CD44 standard isoform and have an EMT phenotype resistant during treatment. Here we determined the role of (CD44s/EMT). In vivo, CD44s/EMT engraft and expand rapidly CD44 expression and its isoforms as a marker and potential and give rise to tumors that express high levels of CD44 isoforms target for tumor cells that give rise to invasive and gemcitabine- that contain multiple exon variants. CD44low-expressing cells resistant tumors. show continued sensitivity to gemcitabine in vivo and knockdown Experimental Design: RT-PCR, Western blotting, and DNA of CD44 in CD44s/EMT cells increases sensitivity to gemcitabine sequencing was used to determine CD44 isoform and expression and decreases invasiveness. levels. Flow cytometry was used to sort cells on the basis of their Conclusions: PDAC cells expressing high levels of CD44s with CD44 expression level. CD44 expression was knocked down a mesenchymal-like phenotype were highly invasive and devel- using shRNA. Tumorigenic properties were determined by clono- oped gemcitabine resistance in vivo. Thus, initial targeting CD44 genic and Matrigel assays, IHC, tumor growth in vivo using or reversing the CD44high phenotype may improve therapeutic luciferase imaging and by tumor weight. response. Clin Cancer Res; 1–13. 2016 AACR.

Introduction (MET), which is believed to favor tumor engraftment and growth of cancer cells at metastatic sites (3, 5, 6). The phrase adaptive Heterogeneity of cancer cells within tumors and established plasticity explains how cancer cells gain selective growth and cancer cell lines is well known. The impact that this heterogeneity survival capabilities through phenotypic changes in response to on pathobiology and therapy has not been fully investigated. their environment. During the last few years, the role of epithelial Epithelial-to-mesenchymal transition (EMT) and acquired cancer cell plasticity in cancer development has gained more attention; stem cell properties are a major cause of cancer cell heterogeneity however, the mechanisms that initiate and enable phenotypic (1, 2). EMT represents an adaptive plasticity defined as loss of changes are not fully defined. membrane polarity and acquired migratory properties that is CD44, a nonkinase transmembrane receptor that binds hya- characteristic of mesenchymal cells (3). EMT is thought to play luronan (HA), has been found to play a role in the EMT process an important role in early cancer cell dissemination and metas- (7, 8). CD44 is a widely expressed adhesion molecule, con- tasis (3, 4). The development of metastasis is believed to require tributing to cell–cell and cell–matrix adhesion, cell growth, EMT, which enables cancer cells to migrate from their primary differentiation, and trafficking, and is highly expressed on stem tumor sites. However, recolonizing at distant sites may require cells(2,9).CD44isencodedby20exonsandundergoes redifferentiation or a mesenchymal-to-epithelial transition extensive alternative splicing to generate CD44 standard (CD44s) and CD44 variant (CD44v) forms. CD44s consists the first five exons (exons 1–5) and last five exons (exons 16– 1 Department of Medicine, Division of Medical Oncology, University of 20). The CD44 variable exons are typically numbered v1 to v10 Texas Health Science Center at San Antonio, San Antonio, Texas. 2Experimental and Developmental Therapeutics Program, Cancer (v1 is not encoded in human) corresponding to the genomic Therapy and Research Center, San Antonio, Texas. 3Department of exons 6–15 and are alternatively spliced and incorporated Pathology, University of Texas Health Science Center at San Antonio, between the exon 5 and exon 16, either as a single exon variant San Antonio, Texas. 4Department of Urology, University of Texas Health Science Center at San Antonio, San Antonio, Texas. 5Research or as a combination of several exons variant (7, 10). Functional and Development, Audie Murphy Veterans Administration Hospital, significance of CD44s and CD44v are not fully understood. San Antonio, Texas. Recent studies show aberrant levels and variant forms of CD44 Corresponding Author: James W. Freeman, Department of Medicine, Division of are expressed in a variety of tumor types including PDAC. High Medical Oncology, University of Texas Health Science Center at San Antonio, level of CD44 is thought to be associated with poor prognosis 7703 Floyd Curl Drive, San Antonio, TX 78229-3900; Phone: 210-567-5298; Fax: ofcancerandCD44v6andv3arereportedtoberelated 210-567-6687; E-mail: [email protected]. to cancer metastasis (9, 11–17). Studies show that CD44v6 doi: 10.1158/1078-0432.CCR-15-3115 expression is largely restricted to the advanced stages of tumor 2016 American Association for Cancer Research. progression and is more prevalent in metastatic than in

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cells may show further plasticity by undergoing a CD44 isoform Translational Relevance change and a mesenchymal-to-epithelial (MET) switch. Chemo- Progression of PDAC after initial response to gemcitabine therapy or other environmental stresses may induce a CD44high treatment is attributed to the expansion of a subpopulation of -expressing cell population that promotes cell survival and inherently resistant tumor cells. It is important to understand increases invasiveness. Thus, targeting CD44high-expressing cells the biology of this resistant subpopulation of PDAC cells and may be an important strategy for increasing the response to to develop strategies for targeting these cells to prevent tumor chemotherapy. regrowth and progression. We found that gemcitabine treatment led to expansion of cells that express CD44. Here we Materials and Methods show the separation of a subpopulation of PDAC cells based Cell culture and materials on high expression level of CD44. This cell population Human pancreatic ductal adenocarcinomas (PDAC) cell lines expressed a high level of CD44 standard isoform, was mes- BxPC-3, Capan-2, MIA PaCa-2, CFPAC-1, Panc-1, AsPC-1 were enchymal-like, highly invasive, and gave rise to gemcitabine- from ATCC, UK Pan-1 was established in our laboratory (29), and resistant tumors that exhibited a CD44 isoform switch with HPNE (hTERT-immortalized human pancreas nestin expressing expression of multi-exon isoform variants of CD44. Knock- cell line) was obtained from Michel M. Ouellette (University of down of CD44 in CD44high cell resulted in cells that were more Nebraska Medical Center, Omaha, NE; ref. 30). Cells were main- sensitive to gemcitabine and less invasive. These studies sug- tained in medium as recommended and supplemented with 10% gest that high CD44 expression is a marker for gemcitabine FBS in a 37C, 5% CO incubator. The plasmid shCD44-2 pRRL resistance in PDAC and that targeting CD44high-expressing 2 was a gift from Bob Weinberg (Addgene plasmid # 19123), which cells may provide a therapeutic strategy for improving survival. targets all isoforms of human CD44 (31). PMMP-luciferase plasmid was from Dr. Ricardo Aguiar (University of Texas Health Science Center, San Antonio, TX). The plasmids were transfected into human embryonic kidney 293T packaging cells (ATCC) using FuGENE 6 (Roche Applied Science) according to the manufac- nonmetastatic colorectal adenocarcinomas (18). Elevated turer's protocol. Cells were then infected with the viral medium expression of CD44v6 in pancreatic cancer appears to play a from the packaging cells 48 hours after transfection. The CD44 functional role in metastasis (13, 19). CD44 isoform switching knockdown cells were selected by GFP cell sorting (Flow Cyto- fromavariantformtoastandardformduringtheEMTprocess metry Core of University of Texas Health Science Center at San has been reported and CD44s is correlated to an EMT pheno- Antonio, TX). The luciferase-expressing clones were selected with type (1, 20, 21). We previously showed that treating a PDAC 300 mg/mL of G418. CD44 levels were determined by Western cell line BxPC-3 with gemcitabine induces cells to undergo an blotting analysis and luciferase expression was determined using EMT, to become more invasive and to show a phenotypic luciferase assay kit (Promega). Gemcitabine-HCl for Injection was switch from expressing mainly low levels of CD44v to expres- manufactured by Eli Lily and was dissolved in sterile saline (0.9% sing high levels of CD44s (22). This EMT/CD44s phenotype sodium chloride) at a concentration of 20 mg/mL. reverses after multiple passages following removal of gemcitabine, suggesting that PDAC cells respond to chemotherapy by FACS and ALDEFLUOR assay adaptive plasticity and that CD44 may play a role in this Subconfluent cells were dissociated with trypsin and washed process. Isoform switching of CD44 may be a part of tumor twice in serum-free medium. Cells were labeled with FITC (fluo- progression and appear to play a role in EMT (23). rescein isothiocyanate) conjugated monoclonal anti-CD44 anti- The expression of CD44s and its isoforms in cancer cells implies body (eBioscience) at 4C for 30 minutes. Brightly and weakly that CD44 plays a role in promoting tumorigenesis, suggesting stained cells were separated as gated (Fig. 1B) by FACS and were that CD44 may be a molecular target for cancer therapy. Cancer designated as CD44high or CD44low cells respectively. The alde- stem cells that express CD44 are reported to be resistant to hyde dehydrogenase (ALDH) activity was determined using an chemotherapy and to give rise to disease progression during ALDEFLUOR assay kit (StemCell Technologies) according to the therapy or recurrent disease following therapy (24–28). The role manufacturer's protocol. Briefly, the cells were stained with ALDE- of CD44 in maintenance of the stem cell phenotype is not fully high FLUOR reagent (bodipy-aminoacetaldehyde) and/or PE (phyco- established. In the current study, we demonstrated that CD44 erythrin)-conjugated monoclonal CD24 antibody (eBioscience) -expressing cells show predominantly a CD44s isoform, display high and incubated for 30 minutes at 37 C. A specific inhibitor of an EMT phenotype, and are highly invasive. CD44 CFPAC-1 ALDH1, DEBA (diemethylamino-benzaldehyde) was used to con- cells showed stem cell properties by forming tumor spheres in high trol background fluorescence. The ALDH activities and positive vitro. Mice implanted with CD44 cells developed rapid tumor high population and CD24-positive population of the cells were ana- growth and metastasis. Tumors from CD44 cells initially lyzed using the BD LSRII analyzer (BD Biosciences). responded to gemcitabine but rapidly developed resistance, whereas tumors from CD44low cells continued to be responsive to gemcitabine for the 22-week treatment schedule. This suggests Reverse transcription PCR that CD44high cells with predominant expression of CD44s have Total RNA from cultured cells or from tumors was extracted by an initial implantation and growth advantage and give rise to TRIzol reagent (Invitrogen) and 1.0 mg of total RNA was reverse gemcitabine-resistant tumors. However, there is an uncoupling of transcribed using the SuperScript III First-Strand Synthesis System CD44s expression and EMT phenotype during tumor expansion (Life Technologies) and 50 ng cDNA was used for the following as these tumors show expression of high levels of CD44 variants PCR reaction. PCR primers used to amplify CD44 isoforms and and E-cadherin. Thus, this population of CD44high-expressing CD44s only have been described previously (13, 32, 33). The

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A C HPNECapan-1Capan-2BxPC-3CFPAC-1AsPC-1Panc-1UK Pan-1MIA PaCa-2 MK Capan-1Capan-2BxPC-3CFPAC-1MIA PaCa-2Panc-1UK Pan-1AsPC-1

CD44v 160 kDa 1.5k CD44s CD44v CD44 v8-10 (seq) 75 kDa 1.0k ZEB1 CD44s 0.5k 1.5k E-Cad 1.0k v3-v6 (seq) 0.5k Vimentin v6 (seq)

1.5k b-Actin 1.0k v6-10 (seq) 0.5k B v6 (seq) Exon 1 2 3 4 5 6 7 8 91011 12 13 14 15 16 17 1819 20 1.1k s1 s3 v4v2s5 v6 v10v8 s7 s9 1.0k CD44v3 s10 0.7k Primer 1 s3 0.5k isoforms Primer 2 v6 s4 0.3k v3 (seq) Primer 3 v6 s8 Primer 4 v3 s8

Primer CD44s s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 CD44s F R ESRP1

D 160 b-Actin 140 120 100 80 60 40 20 10 8 6 4 2 0 Relative CD44s mRNA expression Relative CD44s mRNA

cap-1 BxPC-3 Panc-1 AsPC-3 Capan-2 CFPAC-1 UK Pan-1 Mia PaCa-2

E 60 F 1.2

50 1.0 CD44s CD44s 40 CD44v3 0.8 CD44v3 CD44v6 30 0.6 CD44v6

20 0.4

10 0.2 Relative mRNA expression Relative mRNA

0 expression Relative mRNA 0.0 Capan-1 Capan-2BxPC-3 CFPAC-1 Mia PaCa-2 Panc-1 UK Pan-1 AsPC-1

Figure 1. Association of CD44 isoforms with PDAC cell phenotypes. A, Western blot analysis of a panel of PDAC cell lines for the expression of CD44 and EMT markers, ZEB1, E-cadherin, and vimentin. b-actin was used for the protein loading control. B, the diagrams of PCR primer design for CD44s and CD44v mRNA expression. C, reverse transcription PCR (RT-PCR) analysis of CD44 isoforms and ESRP1 expressed in a panel of PDAC cell lines. CD44v isoforms labeled as CD44v (seq) were conﬁrmed by DNA sequencing. D–F, quantitative RT-PCR with SYBR Green supermix for the expression of CD44s, CD44v3, and CD44v6 DDC containing isoforms. Data represent fold change (mean SD) calculated with 2 t method of triplicate experiments.

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quantitative PCR reaction was done with iQ SYBR Green Super- toneum and liver. Liver metastases were also examined by the mix (Bio-Rad) according to the manufacturer's instruction. The bioluminescent image during the necropsy. CD44s, CD44v3, and CD44v6 primers used for qRT-PCR have DDC been described elsewhere (13, 33). The 2 t method was used Histopathology and IHC for calculating the relative mRNA expression. Paraffin-embedded primary tumor and liver sections were stained with hematoxylin and eosin (H&E) and IHC was per- DNA sequencing formed with mAbs to CD44 (Cell Signaling Technology) or to PCR products from PDAC cell lines or tumors using CD44 Ki67 (Abcam) in the Histology and Pathology Laboratory (Uni- isoform primers were separated by 1.2% agarose gel. DNA frag- versity of Texas Health Science Center, San Antonio, TX). The ments of CD44 isoforms were purified by Zymoclean Gel DNA tumor desmoplasia was evaluated using Sirius Red staining pro- Recovery kit (ZYMO Research) according to the manufacturer's tocol from IHC WORLD, LLC and quantitated using ImageJ instruction. In brief, DNA fragments of our interests were excised software (NIH, Bethesda, MD). The primary tumor and liver by clean scalpel under UV light, agarose gel slices were incubated metastasis were examined under the light microscopy. at 55C for 5 to 10 minutes until they are completely dissolved. Melted agarose was applied to a Zymo-spin column and DNA was Statistical analysis eluted from the column. At least 150 ng of each interested DNA Statistical analysis was performed using GraphPad InStat soft- were sent to the DNA Core Facility in the University of Texas ware (GraphPad Software, Inc.). The significance of differences Health Science Center for DNA sequencing and were then con- among groups was determined by one-way ANOVA followed by fi rmed using the NCBI blast alignment tool. Bonferroni multiple comparison test, t test, or Fisher exact test accordingly. Statistically significance was considered as P < 0.05. Colony and sphere formation assays The tumorigenic properties were determined by colony formation assay and tumor sphere formation assay. For the colony Results formation, single-cell suspension was plated at density of 50, 200, Expression of CD44 and EMT makers in PDAC cell lines and 500 cells per 60-mm dishes and cultured for 2 weeks. The cells A panel of eight PDAC cell lines and HPNE (30), an immor- were washed with PBS, then fixed and stained with 0.1% crystal talized, undifferentiated, nontumorigenic, and nestin-positive violet/40% methanol and colonies 1 mm were counted. For the cell line was examined by Western blotting for the expression of sphere formation, 2,000 cells were seeded per well in a 24-well CD44 and markers of epithelial and mesenchymal phenotypes. low attachment plate (Corning) and cultured in the serum-free The cell lines that express a higher level of CD44 also expressed media containing 2% B27, 1% N2, 20 ng/mL human bFGF greater levels of the mesenchymal marker vimentin, and the EMT- (Invitrogen, Life Technologies) and 50 ng/mL rhEGF (R&D Sys- associated transcription factor ZEB-1 and lower levels of E-cad- tems) for 7 to 10 days. Spheres 30 mm were counted under the herin, an epithelial marker (Fig. 1A). Interestingly, the epithelial- light microscopy. For serial passages, spheres were disassociated like and mesenchymal-like PDAC cell lines express not only to single cell by trypsin and 2,000 cells were replaced per well. different levels, but also distinct patterns of the CD44 standard isoform (CD44s) and variant isoforms of CD44 (CD44v). PDAC Western blotting cell lines and the undifferentiated HPNE cell line that possess a Whole-cell lysates were obtained in Laemmli sample buffer and mesenchymal phenotype express a high level of CD44s and low Western blotting analysis was performed using standard techni- levels of single exon or low molecular size variants of CD44 as ques as described previously (34). Antibodies against CD44 and determined by RT-PCR (Fig. 1C), by DNA sequencing of RT-PCR ZEB1 were purchased from Cell Signaling Technology. E-cadherin products and by quantitative RT-PCR (Fig. 1D and F). In com- and vimentin were purchased from Santa Cruz Biotechnology. parison, PDAC cell lines showing an epithelial-like phenotype expressed CD44v containing combinations of multiple variant Orthotopic pancreatic cancer mouse model exons (Fig. 1A and C). Expression of single exon variant CD44v3 Four to 5-week-old female athymic nude mice were purchased and CD44v6 or low molecular size CD44 isoforms possessing from Harlan Laboratories, Inc. Mice were housed and maintained limited numbers of variant exons were more common to men- in accordance with the standards of Animal Care and Use Com- senchymal-like PDAC cell lines (Fig. 1B, and C, primers 2–4). The mittee of Audie Murphy Veterans Administration Hospital (San relative mRNA levels of CD44s were evaluated by quantitative RT- Antonio, TX). Cells were grown to 80% confluence, trypsinized, PCR for the eight PDAC cell lines (Fig. 1D). In agreement with the and resuspended in saline (0.9% sodium chloride). A total of 2.5 Western blot analysis (Fig. 1A) the mRNA levels of CD44s were 105 cells/50 mL were injected into the tail of the pancreas of higher in the four cell lines that exhibit a clear mesenchymal anesthetized mice. The orthotopic xenograft experiment was phenotype (MIAPaCa-2, Panc-1, UKPan-1, and AsPC-1) com- repeated to determine gemcitabine response in vivo. Two weeks pared with those cells that showed a mixed (CFPAC-1) or an after implantation, mice received either gemcitabine (80 mg/kg) epithelial (Capan-1, Capan-2, BxPC-3) phenotype (Fig. 1D). or saline by intraperitoneal injection, once a week until the end of PDAC cells with an epithelial phenotype showed a relatively experiment. Tumor growth and metastasis were monitored week- lower level of CD44s protein and mRNA expression (Fig. 1A, ly by bioluminescent imaging using the IVIS Imaging system D, and E). Interestingly, the ratio of CD44v (containing v3 and/or (Caliper Life Sciences). Six weeks after implantation, control v6) to CD44s mRNA expression was higher in cells with an group mice showing stress signs were euthanized and remaining epithelial phenotype and the reverse ratio (higher ratio of CD44s mice were continually received gemcitabine once a week until to CD44v) was seen in cells with a mesenchymal phenotype stress signs shown. During necropsy, mice were evaluated grossly (Fig. 1E and F). Correspondingly, ESRP1 was mainly expressed for evidence of tumor invading and metastasizing into the peri- in the epithelial-like phenotype PDAC cell lines (Fig. 1C). Thus,

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A CFPAC-1 CFPAC-1 250 CD44High 200

CD44Low 15010050 High CD44Low CD44 Count SSC-A (x 1,000) 50 100 150 200 250 300 0 102 103 104 105 102 103 104 105 CD44 FITC-A CD44 FITC-A AsPC-1 AsPC-1 Figure 2. Isolation of CD44 high and low cells. CD44High A, cells were labeled with FITC- conjugated monoclonal anti-CD44 High antibody and CD44high and CD44low CD44Low CD44 cells were separated as gated by

FACS. B, light microscopic images Count SSC-A (x 1,000) represent the morphology of 100 150 200 250 CD44high and CD44low cells after 50 cell sorting. C, expression of CD44s, 250 500 750 1,000 CD44Low CD44v, and EMT marker proteins

from cell lines after cell sorting was 0 analyzed by Western blotting. p, 102 103 104 105 102 103 104 105 parental cells; Hi, CD44high cells; CD44 FITC-A CD44 FITC-A Lo, CD44low cells.

BCCD44-Hi CD44-Lo CFPAC-1 AsPC-1 p Hi Lo p Hi Lo

CD44v

CFPAC-1 CD44s

E-cadherin

Vimentin

AsPC-1 b-Actin

PDAC cells with a mesenchymal phenotype express greater total ance and expressed low levels of vimentin and high levels of levels of CD44 with a predominance of CD44s isoform and E-cadherin (Fig. 2B and C). CD44 expression levels were con- CD44v expression was restricted to single variant exon or lesser firmed by Western blotting, which showed that CD44high cells amounts of combination variant isoforms (Fig. 1). expressed higher levels of CD44 with CD44s as the predominant isoform. In comparison with CD44high cells, CD44low cells exp- Isolation of PDAC cells based on expression level of CD44 ressed lower level of CD44s (Fig. 2C). The cell lines CFPAC-1 and AsPC-1 showed low levels of expression of both E-cadherin and vimentin and intermediate Oncogenic properties of CD44high and CD44low cells levels of CD44s with expression of higher molecular weight The oncogenic properties of cells from CD44high and CD44low CD44v isoforms (Fig. 1A), suggesting that these cell lines may populations were compared by determining their clonogenic, represent an intermediate phenotype or contain a mixture of invasive, and tumor sphere–forming capacities. CD44high cells epithelial and mesenchymal phenotypes. To further investigate isolated from CFPAC-1 and AsPC3 parental cells were more these possibilities, CD44high and CD44low expressing populations clonogenic and more invasive than the CD44low cells as deter- of CFPAC-1 and AsPC-1 were separated by flow cytometry mined by colony-forming assay and Matrigel invasion assays (Fig. 2A). The CD44high and CD44low populations displayed (Fig. 3A and B). CD44 is recognized as a cancer stem cell marker different morphologies and expressed differential EMT markers; and is widely used to enrich cancer stem cells in vitro in a variety of CD44high cells were more fibroblast like in appearance and cancers (2, 9). A tumor sphere assay was used to test whether expressed high levels of vimentin and low levels of E-cadherin, CD44 expression and mesenchymal phenotype were correlated whereas CD44low cells exhibit a more cobblestone-like appear- with cancer cell stemness, CD44high-expressing CFPAC-1 cells

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Figure 3. In vitro tumorigenic and invasive properties of CD44high and CD44low cells. A, clonogenic assays were performed as described previously (34). Briefly, cells were seeded at 50, 250, and 500 cells per 6-cm2 dishes and cultured for 2 weeks. The cells were stained with crystal violet and colonies 1 mm were counted. The data were presented as mean SD of experiments performed in triplicates. B, 3 104 cells/well were seeded in 24-well Matrigel invasion chambers for 24 hours. Invaded cells were counted under microscope and the data represent mean SD from triplicate experiments. Top, photos of invasion assays; bottom, quantification data of invasion assays. C, tumor sphere–forming assay of CFPAC-1 cells. Cells (2,000) were plated in a low-attachment 24-well plate and cultured in the medium described in Materials and Methods for 7–10 days and spheres 30 mm were counted. P1–P4 are the passage numbers; every passage lasted for 7—10 days. D, confocal microscopy images of immunofluorescence staining of CD44 and vimentin in tumor spheres of CD44high population cells. CD44-Hi, CD44High; CD44-Lo, CD44Low.

continuously formed spheres through four subsequent serial 3D), whereas, CD44low CFPAC-1–expressing cells show a passages (Fig. 3C) and cells from these spheres express CD44 decreased capacity to form spheres after serial passages (Fig. and vimentin as determined by immunoﬂuorescent staining (Fig. 3C). The current understanding of markers for pancreatic cancer

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stem cells include CD44, CD24, and ALDH1 as reviewed by Vaz the gemcitabine-treated CD44low group of mice through 22 weeks and colleagues. and Medema (35, 36). To further assess stem cell (Fig. 4E and F). properties, CFPAC1/CD44high and CD44low cells were compared To determine whether CD44 expression had an effect on tumor using FACS analyses to detect CD24 and ALDH-positive cells. cell proliferation, immunohistochemical staining for Ki67 was CD24 was detected using an antibody and ALHD bright cells were performed. The results showed that tumor cell proliferation was detected using an ALDEFLUOR assay. CD24þALHD bright or significantly lower in CD44low tumors when treated with gemci- double positive cells were greater in CFPAC-1-CD44high cells tabine, whereas, tumors that grew from the CD44high group (4.3%) compared with CFPAC1-CD44low cells (1.2%; data not following gemcitabine treatment had proliferation rate similar shown). to the saline-treated group (Fig. 5A). To further investigate the role The CD44high population in AsPC-1 cells showed a clear of CD44 in tumor recurrence after gemcitabine treatment, tumor mesenchymal phenotype and lack of E-cadherin; however, the tissues were analyzed by IHC. CD44low tumors showed less CD44low population remained rather intermediate with contin- intensity of staining in saline-treated controls compared with ued higher expression of CD44s than CD44v and intermediate CD44high tumors (Fig. 5B). However, in tumors that grew after levels of E-cadherin and vimentin (Fig. 2C). The lack of sphere- gemcitabine treatment, almost all cells showed intense CD44 forming activity by serial passages in the AsPC-1 high cells (data staining (Fig. 5B). Western blot analysis of tissue from tumors not shown) suggests that expression of CD44s may play a role but derived from CD44high cells showed a switch from expressing is not in itself sufficient for tumor sphere formation and stemness. predominantly CD44s to expressing high levels of CD44 variant isoforms and these tumors also expressed the epithelial marker, E- CD44high/mesenchymal cells are more tumorigenic and are less cadherin (Fig. 5C). The expression of epithelial variant form of responsive to gemcitabine in vivo CD44 v8-10 and ESRP1 in CD44high tumors was confirmed by RT- To compare tumor progression in vivo, we orthotopically PCR analysis (Fig. 5D). This suggests that these rapidly growing implanted either CD44high or CD44low populations of CFPAC- tumors undergo a CD44 isoform switch towards expressing high 1 cells that express firefly luciferase into the pancreas of athymic levels of CD44 variant isoforms and a phenotypic change to a nude mice. Bioluminescent imaging was performed weekly to more epithelial-like tumor cell. An epithelial phenotype may be monitor tumor progression. Six weeks after tumor cell implan- required for the rapid growth of tumor cells. Thus, the greater level tation, mice were euthanized and tumor growth and metastasis of CD44 expression is associated with an increase in tumor were evaluated by tissue examination, tumor weight, and histo- growth; however, the specific roles of the CD44 isoforms in pathology. The quantification data of the bioluminescent imaging engraftment and growth need to be further determined. for the 12 mice in each of the CD44high and CD44low groups is Desmoplasia commonly develops in pancreatic cancer and is shown in Fig. 4A (right) and the representative bioluminescent thought to contribute to chemoresistance. It is possible that CD44 images up to 6 weeks for a representative mouse in the CD44high may play a role in this process. To investigate this possibility, and CD44low groups is shown in Fig. 4A (left). Tumor growth and tumors derived from CD44high and CD44low cells were stained metastatic spread were more rapid in mice implanted with with Sirius Red to detect collagen. No differences of the collagen CD44high-expressing cells. By week 6, mice implanted with staining were found between saline-treated CD44high and CD44high cells developed larger tumors and showed greater local CD44low tumors; however, gemcitabine-treated tumors displayed invasion (Fig. 4A and B) and liver metastasis (Fig. 4C). Biolumi- large area of fibrosis-like pathology and collagen staining nescent imaging of resected livers showed that 9 of 12 mice had (Fig. 5E). This suggests that CD44 expression level does not play metastatic foci in CD44high group while only 2 of 12 mice from the a role in the development of desmoplasia although gemcitabine CD44low group showed positive bioluminescent imaging treatment induces fibrosis perhaps as a result of cell death in (Fig. 4C). Histologically, the metastatic liver foci were within the tumor areas. lymphatic tissue surrounding the liver or showed bile duct involvement with surrounding hepatic invasion (Fig. 4D). Mac- Knockdown CD44 inhibits tumorigenicity and increases roscopic observation also showed evidence of peritoneal metas- sensitivity to gemcitabine tases and tumor attachment to the intestines and by histology of To further look at the role of CD44 in mediating tumorigenic several mesenteric lymph nodes in some mice. properties, CD44 was knocked down in CD44high cells with We next compared the response to gemcitabine for orthotopi- shRNA (Fig. 6F). These studies show that knocking down CD44 cally implanted CD44high and CD44low cells in vivo. Mice were significantly reduced the colony-forming property in CFPAC-1 treated with vehicle (saline) alone or gemcitabine once a week CD44high cells (Fig. 6A) and enhanced their response to gemci- beginning two weeks after implantation. Tumors in the placebo tabine similar to the level of CD44low cells (Fig. 6B). To test our group grew and progressed more rapidly (Fig. 4E and F). These hypothesis that CD44high cells may contain a subpopulation of control groups of mice were euthanized at 6 weeks after implan- cells that possess stem-like properties and are originally resistant tation of tumor cells due to tumor burden and signs of physical to gemcitabine, cells were exposed to high dose of gemcitabine stress in the group implanted with CD44high cells. The gemcita- (50 ng/mL) for 2 weeks. We found that colonies only grew bine-treated group of mice continued to receive weekly treatment. appreciably from the CD44high cells and that knocking down In the first 6 weeks, both CD44high and CD44low groups of mice CD44 from CD44high cells abolished the colony-forming activity responded to gemcitabine and tumor growth was inhibited or in the presence of high dose of gemcitabine (Fig. 6C). The invasive regressed (Fig. 4E). However, in mice treated weekly with gemci- and sphere-forming properties of these cells were tested by in vitro tabine, the CD44high group mice displayed signs of tumor growth Matrigel invasion and sphere-forming assays. Knocking down by week twelve and tumors continued to grow exponentially until CD44 significantly inhibited the invasiveness in both CFPAC-1 the end of the study at week 22 at which time these mice formed and AsPC-1 CD44high cells (Fig. 6D) and prevented the sphere- ascites. Interestingly, tumors remained small to nondetectable in forming activity of CFPAC-1 (Fig. 6E). However, knocking down

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Figure 4. CD44 mediate tumor growth, invasion, and chemoresistance in vivo. A total of 2.5 105 CF/CD44high (CD44-Hi) and CF/CD44low (CD44-Lo) cells were implanted into the tail of pancreas of nude mice. Primary tumor and metastases were examined grossly and pathologically at week 6 after implantation. A, bioluminescent images of tumors from CD44high and CD44low cells monitored weekly by bioluminescent imaging (left). Tumor growth curve plotted from bioluminescent data of CF/CD44high and CF/CD44low groups. The data represent means (n ¼ 12) of each group, Bars, SE. , P < 0.05; , P < 0.01, CF/CD44low versus CF/CD44high group. B, tumors collected at the end of experiment from CF/CD44high and CF/CD44low groups (left) and a quantiﬁcation data of tumor weight of 12 mice each group (right). C, bioluminescent images of liver metastases taken at the end of experiment from CF/CD44high and CF/CD44low mice. D, representative photos of hematoxylin and eosin (H&E)-stained liver sections. The arrows indicate the metastatic foci. E, tumor growth curve plotted from bioluminescent data of CF/CD44high and CF/CD44low groups treated with saline for 6 weeks or gemcitabine for 20 weeks (n ¼ 6 each group). , P < 0.01; , P < 0.001, compared with CF/CD44high group. F, a quantiﬁcation data of tumor weight (mean SE, n ¼ 6 each group). CD44-Hi, CD44High; CD44-Lo, CD44Low; Gem, gemcitabine.

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CD44 and Gemcitabine Resistance

CD44 itself was not sufficient to completely reverse the EMT the standard form and showed an increase in expression of the phenotype of these cells, as the mesenchymal marker, vimentin epithelial marker, E-cadherin, which is expressed at a low level or expression was only slightly reduced and the epithelial marker, E- not present in the original CD44high cells. This phenomenon cadherin was not reexpressed in the CD44 knockdown clones suggests that cells from CD44high-induced tumors undergo MET (Fig. 6F). and that a CD44 isoform switch may be required for this to occur. As these tumors appear to be resistant to gemcitabine, this suggests Discussion an uncoupling of CD44s expression and gemcitabine resistance. However, these tumors continue to express high levels of CD44 but In this study, we show that the phenotypic and tumorigenic as variant isoforms. Thus, the level of CD44 may be important in properties of PDAC cells are related to the level of expression and maintaining resistance. Desmoplasia an important component of type of CD44 isoform. An initial observation from analyses of a PDAC (37, 38) did not appear to differ appreciably in tumors panel of PDAC cell lines was that cells showing an EMT phenotype grown from CD44high or CD44low cells but was dramatically express higher levels of CD44 with a predominance of the stan- increased by gemcitabine treatment. dard isoform. To further look at heterogeneity of CD44 expres- EMT and MET are dynamic processes in tumor progression, and sion, we sorted out CD44high and CD44low-expressing cells from CD44 may play a role in regulating these processes. EMT is two separate PDAC cell lines, CFPAC-1 and AsPC-1. CD44high involved in invasion, tumor metastasis, and stem cell properties, cells express predominantly a CD44s isoform, display an EMT whereas, conversion back to an epithelial phenotype may favor phenotype, and are more invasive in vitro. In comparison, tumor growth (39). Tumor cells once engrafted, may need CD44v CD44low cells express relative low level of CD44s and low levels to convert EMT-like cells to epithelial-like cells (MET); otherwise, of larger size CD44 variants, are more epithelial like, and are less CD44s may be required to facilitate cells to undergo EMT, which invasive. Our finding that PDAC cells with an EMT phenotype increases local invasion and for metastasizing to the distal regions. express mainly CD44s isoform is in agreement with a study by Thus, the total level of CD44 as well as the isoform may be Brown and colleagues (20) showing that induction of EMT in important in tumor progression. EMT appears in this study to be HMLE cells was associated with a switch from expressing mainly closely associated with loss of ESRP1 and a decrease in expression CD44v isoforms to expressing predominantly CD44s. Our study of CD44 multi-exon variants. ESRP1 has been reported to play a is also in agreement with that of Mani and colleagues (1) showing role in the isoform switch of CD44 (6, 20, 21, 40). Expressing that EMT was associated with higher level in expression of CD44. ESRP1 in PDAC cells was shown to decrease proliferation, inva- Interestingly, the study by Mani and colleagues (1) shows that sion, and metastases (41) We showed here that ESRP1 expression induction of EMT generates cells that have properties of stem cells. was associated with cells that are epithelial-like and express lower Here we also show using the CFPAC-1 model that CD44high but levels of total CD44, of which most express CD44v molecules that not CD44low-expressing cells were able to grow in serial passages possess multiple variant exons. ESRP1 expression was decreased as tumor spheres. However, only a low percentage of cells (4.3%) or lost in the mesenchymal-like cells that mainly express CD44s. from the CD44high group show coexpression of other stem cell We also demonstrated that the tumors derived from CD44high markers including CD24 and ALDH. To this point, knockdown of cells underwent CD44 isoform switch and gained the ESRP1 CD44 in CD44high cells was sufficient to prevent growth of these expression when grown as orthotopic implants. This observation cells as tumor spheres. However, CD44high cells from ASPC-1 cells is consistent with the premise that the molecular basis of CD44 were not able to form tumor spheres. These results suggests that isoform switch is regulated by ESRP1. Therefore, regulation of CD44 may play a role but is not sufficient by itself for a stem cell ESRP1 expression likely controls, at least in part, the type of CD44 phenotype. isoform expressed and downregulation of ESRP1 likely facilitates In addition to expressing high levels of CD44s, PDAC cells with expression of mainly the form of CD44s. an EMT phenotype express small molecular size exon variant CD44 is highly expressed in variety of tumors, tumor-initiating isoforms of CD44v3 or CD44v6. This phenomenon appears cells, and recurrent tumors, suggesting that it could be a thera- unique for cells with a mesenchymal phenotype as cells with an peutic target for cancer. Continued progression and recurrence of epithelial phenotype express CD44v isoforms that contain pre- cancers following chemotherapy is thought to be due to inherent dominantly multiple variant exons. A number of studies suggest or acquired resistance to the therapeutic agent. In this study, we that expression of CD44 variants including CD44v3 and v6 are treated the CD44high and CD44low tumor-bearing mice with associated with metastatic lesions (16, 17, 19, 32). The functional gemcitabine. Although both CD44high and CD44low tumors differences of CD44 molecules that possess single or multiple responded to gemcitabine at the beginning, CD44high tumors variant exons is not clear and needs further investigation. The high gradually develop resistance after 12 weeks of treatment. CD44low level of expression in CD44high cells suggests that CD44s isoform tumors continued to be sensitive to gemcitabine through 22 plays an important role in tumor cell plasticity contributing to weeks suggesting that CD44 expression level is related to che- increased invasiveness. However, this study does not rule out the moresistance and disease progression. We have also demonstrat- possibility that minor variant isoforms contribute significantly to ed that knocking down CD44 in CD44high cells attenuated col- these properties. ony-forming activity and invasiveness and increased the sensitiv- The functional significance of the CD44 in CFPAC-1 cells was ity to gemcitabine. A subpopulation of CD44high cells may possess further assessed using an orthotopic mouse model. Tumors devel- stem cell properties that are inherently resistant to gemcitabine. oped from CD44s/EMT cells grew and metastasized more rapidly Preclinical studies show promising results by targeting CD44 than CD44low expressing cells. However, once tumors grew in vivo, using CD44 antibodies (24, 27). These studies demonstrated that the tumor cells underwent a phenotypic change. Western blot and relapsing tumors are sensitive to anti-CD44 treatment; therefore, RT-PCR analyses show that tumor specimens from the CD44high targeting CD44 could have a therapeutic benefit. Our study group express higher level of CD44 variant isoforms (v8–10) than suggests the possibility that targeting the CD44high-expressing

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Figure 5. A, representative pictures of immunostaining of tumor sections with mAb to Ki67 (left) and quantification data of Ki67-stained tumor sections analyzed by ImmunoRatio online software (right). The data were presented as the mean SD. B, representative pictures of IHC of tumor sections with CD44 mAb. C, tumor tissue lysates from saline-treated group and collected 6 weeks after implantation. Fifty micrograms of the tissue lysates were analyzed by Western blot analysis with indicated antibodies. b-actin was used for the protein loading control. Last two lanes labeled as "Cell" are lysates from CF/CD44high and CD44low cells used for the implantation. D, RT-PCR analysis of tumor tissues treated with saline using the primer 1 as indicated in Fig. 1B and CD44 isoforms were separated in 1.2 % agarose gel. CD44v isoforms labeled as CD44 v8–10 (seq) were confirmed by DNA sequencing. E, representative photos of Sirius Red staining of tumor sections (left) and a quantification data (right) analyzed using ImageJ program (NIH, Bethesda, MD). CD44-Hi, CD44High; CD44-Lo, CD44Low; Gem, gemcitabine.

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Figure 6. A and B, 500 cells were plated per 6-cm2 dishes. The next day, cells were treated with one dose of 0, 2.0, and 5.0 ng/mL gemcitabine and cultured for 2 weeks. The cells were stained with crystal violet and colonies 1 mm were counted. A, the data were presented as mean SD of experiments performed in triplicates. B, the data were calculated as the percent of colony number relative to control (gemcitabine 0 dose). C, 5,000 cells were plated per 6-cm2 dishes. The next day, cells were treated with gemcitabine (50 ng/mL) and gemcitabine was replenished every week for 2 weeks. Colonies 1 mm were counted. The data represent the mean SD for triplicate experiments. D, 3 104/well cells were seeded in 24-well Matrigel invasion chambers for 24 hours. Invaded cells were counted under microscope and the data represent mean SD from triplicate experiments. , P < 0.05; , P < 0.01; and , P < 0.001 compared with CD44high cells. E, tumor sphere–forming assay of CF/CD44high, CF/CD44low, and shCD44 cells. Cells (2,000) were plated in a low-attachment 24-well plate and cultured in the medium described in Materials and Methods and passaged every 7–10 days and then the 2,000 cells were replated. The passages–4 spheres ( 30 mm) were counted. F, CD44 levels and EMT marker proteins in CD44 knocking down CF/CD44high and As/CD44high cells analyzed by Western blotting. cl.1 and cl.2 represents shCD44 clone 1 and clone 2, respectively.

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Zhao et al.

tumor cells may increase and prolong response to therapy and Authors' Contributions perhaps prevent the outgrowth of a gemcitabine-resistant popu- Conception and design: A.P. Kumar, J.W. Freeman lation. In our studies, CD44high cells formed tumors that gradually Development of methodology: S. Zhao developed resistance to gemcitabine. It is of interest that the Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Zhao, C. Chen, K.W. Chang, J.W. Freeman gemcitabine-resistant tumors continued to express high total Analysis and interpretation of data (e.g., statistical analysis, biostatistics, levels of CD44 composed of CD44s but also showed a dramatic computational analysis): S. Zhao, C. Chen, K.W. Chang, J.W. Freeman increase in the levels of CD44v that possess multiple variant CD44 Writing, review, and/or revision of the manuscript: S. Zhao, C. Chen, exons. K.W. Chang, A. Karnad, A.P. Kumar In summary, PDAC cell lines show heterogeneity in regards to Administrative, technical, or material support (i.e., reporting or organizing CD44 isoform and level of expression. In vitro, CD44high-expres- data, constructing databases): S. Zhao Study supervision: A.P. Kumar, J.W. Freeman sing cells show predominantly a CD44s isoform, display an EMT Other (review of pathology slides): J. Jagirdar phenotype, and in vivo are highly invasive and form tumors and high metastases more rapidly. Mice implanted with CD44 cells Acknowledgments show initial response to gemcitabine but gradually show rapid We thank the Flow Cytometry Core Facility and the Histology and expansion and loss of response to gemcitabine. Mice implanted Pathology Laboratory (University of Texas Health Science Center, San with CD44low cells showed a continued response to gemcitabine Antonio, TX). over a 22-week period. This suggests that CD44low-expressing phenotype will have a prolonged response to chemotherapy. Grant Support high Molecular analyses show that CD44 tumors continue to This work was supported by grants from the William and Ella Owens Medical express high levels of CD44 but with a switch to expressing Research Foundation, VA Merit Award, Cancer Center Grant to CTRC and NIH- increased levels of CD44v isoforms. Knockdown of CD44 in P30CA054174 (to J. W. Freeman), and CPRIT training grant (to K. Chang). CD44high cells causes these cells to be less invasive and more The costs of publication of this article were defrayed in part by the sensitive to gemcitabine. This study suggests that targeting CD44 payment of page charges. This article must therefore be hereby marked high ﬁ advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate in PDAC that show a CD44 expression level may bene t this fact. therapy and improve overall response.

Disclosure of Potential Conﬂicts of Interest Received December 31, 2015; revised April 6, 2016; accepted May 10, 2016; No potential conﬂicts of interest were disclosed. published OnlineFirst June 7, 2016.

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CD44 Expression Level and Isoform Contributes to Pancreatic Cancer Cell Plasticity, Invasiveness, and Response to Therapy

Shujie Zhao, Chen Chen, Katherine Chang, et al.

Clin Cancer Res Published OnlineFirst June 7, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-15-3115

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