Effects of adjuvant chemotherapy on bone marrow mesenchymal stem cells of colorectal cancer patients

Effects of adjuvant chemotherapy on bone marrow mesenchymalstem cells of colorectal cancer patientsJ.Cao *,M.H.Tan,P.Yang,W.L.Li,J.Xia,H.Du,W.B.Tang,H.Wang,X.W.Chen,H.Q.XiaoDepartment of Gastrointestinal Surgery,Affiliated Guangzhou First Municipal People’s Hospital,Guangzhou Medical College,1#Pan Fu Road,Guangzhou,Guangdong 510180,ChinaReceived 31October 2007;received in revised form 25December 2007;accepted 2January 2008AbstractBackground.Chemotherapy damages the bone marrow and that is one of the most important problems in the treatment of malignancies,particularly colorectal cancer.The aim of the present study was to assess the effects of surgical adjuvant chemotherapy for CRC patients on human MSCs using an in vitro culture system.Methods.The bone marrows of 43CRC patients were harvested for separation and culture of MSC at pre-and post-chemotherapy.The number of colonies form-ing unit-fibroblast (CFU-F)was counted.The adhesive function of MSC was assayed and the growth of colony-forming unit-mixed hematopoietic cell (CFU-Mix)on the MSC layer was observed.The concentration of IL-6,SCF and FLT-T3proteins in the MSC culture supernatant were also detected by ELISA assay.Results.In the CRC patients with chemo-therapy,we have demonstrated that the CFU-F exhibit significantly decreased.We also showed that the adhesive rate of bone marrow mesenchymal stem cell (BMSC)was significantly decreased.The growth of CFU-Mix on the MSC layer was inhibited.Most importantly,decreased CFU-F and the adhesive rate of BMSC were correlated significantly with decreased interleukins and stem-cell factor (IL-6,SCF and FLT-3L)expressions in the CRC patients after chemotherapy.Conclusion.Our results suggest that MSCs of CRC patients can be damaged by chemotherapy.Our data also indicates that the decreased expression of haematogenesis factors may play an important role in the pathogenesis.In the future,the MSC refused may have a potential clinical application in chemotherapeutically treated patients.Ó2008Elsevier Ireland Ltd.All rights reserved.Keywords:Chemotherapy;Bone marrow mesenchymal stem cells (BMSC);Colorectal cancer;Apoptosis1.IntroductionColorectal cancer (CRC)is the third most com-mon type of cancer and the second leading causeof cancer-related death in North America and China.Although surgical resection alone is poten-tially curative,local or distant recurrences develop in many patients,and those with the highest risk of recurrence are advised to receive systemic adjuvant chemotherapy,which has been shown to be beneficial in a number of trials and analyses [13].At present,many studies have shown that0304-3835/$-see front matter Ó2008Elsevier Ireland Ltd.All rights reserved.doi:10.1016/j.canlet.2008.01.011*Corresponding author.Tel.:+862081048888;fax:+862081048000.E-mail address:czhongt@ (J.Cao).Available online at Cancer Letters 263(2008)197–203/locate/canletchemotherapy and/or radiation therapy damage the bone marrow(BM)microenvironment and may result in diminished or delayed hematopoiesis[4]. Therefore,chemotherapy damages the BM and that is one of the most important problems in the treat-ment of malignancies,particularly CRC.Recently, increasing evidences suggest that the damaged hematopoietic microenvironment(HM)must recon-stitute itself to provide the optimal environment for hematopoietic regeneration by mesenchymal stem cells(MSCs)[12].However,the effects of MSCs, the precursor cells of the bone marrow stromal cells are poorly understood.MSCs are important cellular components of the bone marrow microenvironment for supporting haemopoiesis.MSCs produce adventitial cells in the marrow microenvironment;these cells provide support to hematopoiesis by producing mem-brane-bound and soluble signals and cytokines. MSCs constitutively secrete interleukins(ILs),and stem-cell factor,and they are inducible with IL-1a and leukemia-inhibiting factor[10].Therefore,we postulated that MSCs may enhance hematopoietic engraftment rate and quality after myeloablative and stroma-damaging treatments.In addition to the haemopoietic stem cells(HSCs),human bone marrow contains a second type of stem cells, recently termed mesenchymal stem cells(MSCs). MSCs represent an important cellular component of the bone marrow microenvironment in support-ing haemopoiesis[10,11,3].However,their response to surgical adjuvant chemotherapy remains unknown.Recent studies expressed concerns that both chemotherapy and radiotherapy,as used in the conditioning before bone marrow transplanta-tion(BMT),have persistent serious effects on bone marrow MSCs and thus potentially impair haemo-poiesis[9].Previous studies evaluated the number of MSCs and the capacity of stromal layers in supporting allogeneic haemopoietic progenitors [3].In addition,MSCs are progenitors of mesenchy-mal tissues,such as bone,cartilage,muscle,liga-ment,tendon,and adipose.MSC damage may therefore contribute to some mesenchymal tissue diseases,such as osteopenia[6,1].Knowing how to preserve or destroy MSCs is necessary in clinical practice.It is therefore important to understand the MSCs damage pattern caused by chemotherapy. The aim of the present study was to assess the effects of surgical adjuvant chemotherapy for CRC patients on human MSCs using an in vitro culture system.2.Materials and methods2.1.PatientsBetween October2005and October2006,43patients with locally advanced or metastatic colorectal cancer who were eligible for surgical adjuvant chemotherapy in the Department of Gastrointestinal Surgery,Affiliated Guangzhou First People’s Hospital,Guangzhou Medical College.Patients with histologically and/or cytologically proven,locally advanced colorectal cancer and/or meta-static disease were enrolled in this study.The clinical trial protocol and the consent form were approved by the Insti-tutional Review Board for Human Investigation of the Affiliated Guangzhou First People’s Hospital.Chemotherapy was administered concurrently post-operation,using5-fluorouracil(5-FU)450mg/m2/day given as continuous intravenous infusion,and cisplatin (CDDP)2mg/m2given as30-min infusion.All patients were treated with intravenous infusion therapy of5-FU and CDDP for5days,and were treated for6cycles.They were required to have a World Health Organization (WHO)performance status of0–1,and adequate func-tions of bone marrow(WBC count>4000/l l,platelet count>100,000/l l,hemoglobin>10g/l l),renal(serum creatinine concentration<1.5mg/dl,blood urea nitrogen <20mg%)and hepatic functions(serum transaminase level<2Âof the upper normal range).As a control,10 normal health individuals were also obtained in this study.2.2.Collection and isolation of MSCThe collection,isolation,purification and expansion of bone marrow MSCs were conducted by the method reported by Pittenger et al.[14].Briefly,5ml of heparin-anticoagulated bone marrowfluid from43CRC patients between pre-and post-adjuvant chemotherapy was taken and mononucleated cells were isolated by density gradient fractionation(below1.073g/ml,Pharmacia,USA),which were seeded into a75cm2cultureflask in Dulbecco’s modified Eagle’s medium(DMEM)-LG containing10% fetal bovine serum,and cultured at37°C and5%CO2 for primary culture.MSCs of the concentrations of 1Â105and1Â104,were added into the culturefluid of the mixed PBLs to be co-cultivated for5days.At80% confluence,the cells were passaged.2.3.Immunophenotyping of cultured MSCsMSCs(2Â105cells)were incubated with anti-CD29, anti-CD34,anti-CD44,anti-CD45,anti-CD73,anti-CD105,anti-CD166,anti-HLA-A/B/C and anti-HLA-DR(Phar Minge,USA)at room temperature for 30min,respectively.After washing twice with phosphate buffer saline(PBS),the cells were suspended in PBS and198J.Cao et al./Cancer Letters263(2008)197–203analyzed on aflow cytometer(FACS Calibur).MSCs were incubated with the FITC-labeled secondary antibody avoiding light for15.2.4.Measurement of the adhesive rate of MSCsAfter4weeks cultured,the adhesive capacity of MSCs was measured by determining the adhesive rate of MSCs from43patients.MSCs(4Â105cells per well)were pla-ted in24-well plates in a volume of1ml of MethoCult GF H4434(Stem Cell Technologies,Vancouver,BC,Canada) consisting of20%fetal bovine serum(FBS).After2h cul-tures were washed two times with PBS,and collected and counted non-adhesive cells.We analyzed the adhesive rate of MSCs following the formula:The adhesive rate of MSCs=(1Ànon-adhesive MSCs/4Â105)Â100%.2.5.Cell proliferation and colony-forming unit-fibroblast (CFU-F)assaysCell proliferation of MSCs was analyzed by CFU-F assay.CFU-F assay was performed to evaluate the colo-nal expansion capacity of MSCs from43patients.To allow for optimal counting of the colonies,MSCs (4Â105cells per well)were plated in24-well plates in a volume of1ml of MethoCult GF H4434(Stem Cell Tech-nologies,Vancouver,BC,Canada)consisting of30%fetal bovine serum(FBS),2mM L-glutamine,10À4M2-mercaptoethanol,and0.9%methylcellulose.The colony-forming unit-fibroblast of MSCs(colony-forming unit in culture[CFU-F])was assayed as described previously [5].After14days,the cultures were washed three times with PBS/def(pH7.2–7.3)fixed with acetone–methanol for15min(3:2parts vol/vol,0°C;Merck KGa,Darms-tadt,Germany),air-dried,and rehydrated for10min with deionized water for Harris’hematoxylin staining(10–12min;Merck).Colonies consisting of a minimal cell number of50cells were counted under a microscope. 2.6.The growth of colony-forming unit-mixed hematopoietic cells(CFU-Mix)on the MSC layer assay MSCs were trypsinized.Afinal concentration of 4Â105cells/mL was irradiated.Single dose of15Gy, respectively,was administrated at the total of60Gy using a10MV photon(Varian,Palo Alto,CA,USA).Cells from43patients were irradiated in suspension instead of monolayer to avoid subjecting irradiated cells to further stress of manipulation such as trypsinization after irradi-ation which may interfere with cell recovery.To ensure adequate build up and homogenous irradiation,the spec-imen tubes were placed in the center of a tissue equivalent, 10Â25Â11.5cm(widthÂlengthÂheight)perspex box. Radiation was delivered with lateral opposingfields of 10Â20cm to the perspex box housing the specimen tubes and calibration of dose was performed before experi-ments.During the time of irradiation,the control samples were kept outside the machine at the same temperature as the irradiated cultures.The control cells and irradiated cells were plated atflat bottom96-well plate in afinal vol-ume of100-l l culture medium per well.Then after7days, the growth of CFU-Mix on the MSC layer was assessed by microscopy.2.7.Assays of cytokineBecause cytokines are known to be an important growth and survival factor for bone marrow cells,we hypothesized that IL-6,SCF and Flt-3L would be the likely candidate for the growth-promoting component of MSC-CM.Multiplex human cytokine detection(Upstate, Waltham,MA)was utilized to measure the production of interleukin(IL)-6,SCF and Flt-3L in one50-l l aliquot of the supernatant in the cultured MSCs(4Â105cells per well)between pre-and post-adjuvant chemotherapy. Human IL-6,SCF and Flt-3produced in cell supernatant was quantified using the human IL-6ELI-PAIRS kit, SCF ELI-R&D and Flt-3ELI-R&D Kit in per well (4Â105cells),according to the manufacturer’s recom-mendations(Diaclone,Besanon,France).Quantification was performed at least three times.2.8.Statistical analysisThe SPSS statistical analysis software was used for sta-tistical analysis.Two-way analysis of variance was used for the comparison of cell growth and chemotherapeutic sensitivity.P value<0.05was considered statistically significant.3.Results3.1.Patient characteristicsThere were25male and18female patients with an average age of56.2and a range from46to67years. Among the43cases,10,11,14and8were classified as Dukes’stage A,B,C and D,respectively(Table1).3.2.Cell morphology of MSCsHuman bone marrow–derived MSCs were successfully isolated from bone marrow aspirate from43CRC patients and10normal healthy individuals,and all were successfully culture-expanded.A morphologically homog-enous population offibroblast-like MSCs was detected in pre-adjuvant chemotherapy(Fig.1B),which reached greater than90%confluence in a median of14days.There was a good correlation between the number of mononu-clear cells plated to initiate MSC cultures and the number of MSCs recovered.The cell population and morphology of MSCs with pre-chemotherapy is similarly observed inJ.Cao et al./Cancer Letters263(2008)197–203199the MSCs from normal healthy individuals (Fig.1A).However,under standard culture conditions,the size and the cell population of fibroblast-like MSCs were decreased at the post-adjuvant chemotherapy (Fig.1C).Our results also showed that there were not correlation between the differentiation and stages of CRC and the cell size and the population of MSCs.3.3.Immunophenotyping of cultured MSCsWe measured the cellular surface markers of MSCs by FACS.Our results only present that CD4expression was significant decreased in MSCs at the post-chemotherapy compared with the pre-chemotherapy (Table 2).However,the expression of the other surface markers did not identify significant decreased at the post-chemotherapy (Table 2).3.4.Measurement of the adhesive rate of MSCsWe measured the adhesive capacity of MSCs in the CRC patients at the pre and post-adjuvant chemotherapy by determining the adhesive rate of MSCs.The adhesive rate of MSCs at the pre and the post-adjuvant chemother-apy were 80.2±5.7and 53.9±5.1,respectively (Table 3).In normal controls,the adhesive rate of MSCs was 81.4±6.5(Table 3).The adhesive rate of MSCs was sig-nificant decreased at the post-adjuvant chemotherapy compared with the pre-adjuvant chemotherapy (t =3.11,P <0.01)(Table 3).However,the adhesive rate of MSCs was similarly observed in hMSCs at pre-adjuvant chemo-therapy and normal healthy individuals (t =0.22,P >0.05)(Table 3).3.5.Cell proliferation and colony-forming unit-fibroblast (CFU-F)assays of MSCsTo determine whether adjuvant chemotherapy affected the proliferation function of MSCs,we analyzed the hMSC phenotype and the cell proliferation capacity inTable 2The expression of cellular surface markers of MSCs at the pre-and post-chemotherapyCD29CD34CD44CD45CD73CD105CD166HLA-A/B/C HLA-DR Pre-chemotherapy 86.15 1.4889.56 6.6984.7287.2483.4288.15 2.13Post-chemotherapy85.310.7264.84a4.3580.0985.7479.2585.231.86aP <0.05.Fig. 1.The cell morphodifferentiation of MSCs was detected by microscopy.The population and morphology of MSCs with pre-chemotherapy (B)is similarly observed in the MSCs from normal healthy individuals (A).The size and the cell population of fibroblast-like MSCs were decreased at the post-adjuvant chemotherapy (C).Table 1The patient’s clinical and pathological characteristicsCRC casesControl individuals Age >50246<50194Sex M 255F185Differentiation Well 22Median 11Poor 10Dukes’A 10B 11C 14D8Table 3The adhesive rate of MSCs at the pre-and post-adjuvant chemotherapyThe adhesive rate of MSCst ValueP ValueNormal control group 81.4±6.5Pre-chemotherapy group 80.2±5.70.22P >0.05Post-chemotherapy group53.9±5.13.11P <0.01200J.Cao et al./Cancer Letters 263(2008)197–203the CRC patients at the pre and post-adjuvant chemother-apy by CFU-F assay.The MSCs’proliferation was shown in Fig.1.The results of CFU-F of MSCs at the pre-and post-adjuvant chemotherapy were 20.7±2.3/4Â104MNC and 14.8±1.5/4Â104MNC,respectively (Table 4).In normal controls,the result of CFU-F of MSCs was 21.5±3.1/4Â104MNC.The CFU-F of MSCs was significant decreased at the post-adjuvant chemotherapy compared with the pre-adjuvant chemotherapy (t =3.02,P <0.01)(Table 3).However,the CFU-F was similarly observed in hMSCs at pre-adjuvant chemotherapy and normal healthy individuals (t =0.25,P >0.05)(Table 4).3.6.The growth of CFU-Mix on the MSC layer assay After a 7-day cultures,the growth of the CFU-Mix on the MSC layer was assessed by microscopy.The growth of CFU-Mix on the MSC layer was observed in Fig.2.Our results showed that the CFU-Mix on the MSC layer pre-sented smaller and lower concentration at the post-adju-vant chemotherapy group (Fig.2C).At the pre-adjuvant chemotherapy group,the CFU-Mix on the MSC layer presented larger and higher concentration (Fig.2B).The CFU-Mix on the MSC layer was similarly observed at pre-adjuvant chemotherapy (Fig.2B)and normal healthy individuals (Fig.2A).3.7.Assays of cytokineWe examined the levels of IL-6,SCF and Flt-3L from the supernatant in the cultured MSCs between pre-and post-adjuvant chemotherapy by ELISA.We measuredthat the expression levels of IL-6,SCF and FLT-3were 114.25±27.8,1917±415and 831±175at the post-adju-vant chemotherapy group,158.92±33.4,3005±586and 1289±259at the pre-adjuvant chemotherapy group,164.83±36.5,3039±614and 1317±296at normal con-trol group,respectively.Our results identified that MSCs cultured from post-adjuvant chemotherapy expressed low levels of IL-6,SCF and Flt-3L message compared with MSCs from both pre-adjuvant chemotherapy and normal healthy individuals (Table 5).4.DiscussionCurrent literatures indicate that adjuvant chemo-therapy offers advanced CRC patients a significant increase in survival.However,various groups reported that adjuvant chemotherapy damage the bone marrow (BM)microenvironment and may result in diminished or delayed hematopoiesis [9].The delayed hematopoiesis results to the serious complication of immune deficiency,infection and hemorrhage.Recently,many investigators reported that MSCs represent an important cellular compo-nent of the bone marrow microenvironment in supporting haemopoiesis [14,15].MSCs,as the pre-cursor cells of the bone marrow stromal cell,have the characteristics of self-proliferation and multidif-Table 4CFU-F of MSCs at the pre-and post-adjuvant chemotherapyCFU-F of MSCst ValueP ValueNormal control group 21.5±3.1/4Â105MNCPre-chemotherapy group20.7±2.3/4Â105MNC0.25P >0.05Post-chemotherapy group14.8±1.5/4Â105MNC3.02P <0.01Fig.2.The growth of CFU-Mix on the MSC layer was demonstrated by microscopy.CFU-Mix on the MSC layer presented smaller and lower concentration at the post-adjuvant chemotherapy group (C).The CFU-Mix on the MSC layer was similarly observed larger and higher concentration at pre-adjuvant chemotherapy (B)and normal healthy individuals (A).Table 5The expression levels of IL-6,SCF and FLT-3at the pre-and post-adjuvant chemotherapyIL-6SCF FLT-3L Normal control group164.83±36.53039±6141317±296Pre-chemotherapy group158.92±33.4a 3005±586a 1289±259a Post-chemotherapy group114.25±27.8b 1917±415b831±175ba P >0.05,pre-chemotherapy group vs normal control group.bP <0.01,post-chemotherapy group vs pre-chemotherapy group.J.Cao et al./Cancer Letters 263(2008)197–203201ferentiation,and can produce many kinds of hema-topoietic regulating factors,which play an impor-tant role in normal hematopoiesis[8].However, Jager et al.,reported‘‘the survival of the mesenchy-mal progenitor cells in bone marrow during COSS-96polychemotherapy,which allows for an osteogenic regeneration in vitro and potentially in vivo”[7].Therefore,it is important to further explore the function of MSCs in the CRC patients at the post-surgical adjuvant chemotherapy.In our present study,we demonstrated that the size and the cell population offibroblast-like MSCs exhib-ited significantly decreased at the post-adjuvant chemotherapy compared with pre-adjuvant chemo-therapy(P<0.01)(Fig.1,Table4).Additionally, the adhesive rate of MSCs was significant decreased at the post-adjuvant chemotherapy compared with the pre-adjuvant chemotherapy(t=3.11,P<0.01) (Table3).The adhesive rate of MSCs was similarly observed in hMSCs at pre-chemotherapy and nor-mal healthy individuals(t=0.22,P>0.05)(Table 3).This demonstration of the essential role of stro-mal support in hematopoiesis shaped the view that cell–cell adhesion in the bone marrow microenvi-ronment is necessary for normal hematopoietic function and differentiation.Furthermore,we explored the molecular mecha-nism of decreased MSCs in CRC patients at post-adjuvant chemotherapy.Recently,the investigators reported the expression of many growth factor activities associated with hematopoietic support by expended mesenchymal stromal cultures[2].In pres-ent study,we assayed the expression of chemokines in the MSCs cultures at pre and post-adjuvant che-motherapy.We demonstrated that MSCs cultured from post-adjuvant chemotherapy expressed low levels of IL-6,SCF and Flt-3L message compared with MSCs from both pre-adjuvant chemotherapy and normal healthy individuals(Table5).Our results provided critical evidence that the prolifera-tion of mesenchymal stem cells is dependent on sol-uble factors including IL-6,SCF and Flt-3L.In summary,we demonstrated that the function of bone marrow can be inhibited in the CRC patients following standard adjuvant chemother-apy.Our results indicate the potential mechanism leading to adjuvant chemotherapy-induced inhibi-tion of the function of bone marrow could involve the function and the differentiation of mesenchymal stem cells damage caused by soluble factors includ-ing IL-6,SCF and Flt-3L following adjuvant che-motherapy administration.In the future,the MSC refused may be has a potential clinical application in chemotherapeutically treated patients. 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