colorectal cancer ( crc ) is one of the most prevalent cancer , along with lung and breast cancers . in 2012 ,
approximately 1.4 million new cases and 700,000 crc - related deaths were estimated worldwide ( 1 ) . over the last decade ,
crc incidence and mortality in the united states have been on a steady decline as a result of advances in its prevention , screening , and treatment .
nevertheless , it is still the second leading cause of cancer deaths ( 2 ) .
moreover , there has been a significant rise in crc incidence in newly developed or economically transitioning countries , such as south america , eastern european countries , and most parts of asia ( 3 ) .
although crc is highly curable at an early stage , approximately 50% of patients with crcs eventually develop metastatic disease ( 4 ) .
five - year survival rate is approximately 10% in patients with a stage iv disease ( 5 ) .
there are many known risk factors for crc including age , sex , smoking , excessive alcohol consumption , high intake of red and processed meat , obesity , diabetes , inflammatory bowel disease , inherited genetic disorder , and family history of crc ( 6789101112 ) . among them ,
ulcerative colitis and crohn 's disease  increase the overall risk of colitis - associated cancer ( 14 ) .
approximately 20% of patients with inflammatory bowel disease develop colitis - associated cancer within 30 years of disease onset ( 15 ) . inherited crc , including lynch syndrome and familial adenomatous polyposis , represent less than 5% of crc cases ( 16 ) . in this review ,
we briefly review currently available drugs for the treatment of colorectal cancer and summarize clinical studies that use cell - based cancer immunotherapy as promising therapeutic strategy .
we also suggest the use of cytokine - induced killer ( cik ) cells as an additional therapeutic approach for metastatic colorectal cancer .
most crcs occur without a known genetic risk factor and significant family history . only a small proportion ( 5% ) of inherited crcs , which is also known as familial crc ,
have been thoroughly characterized ( 17 ) , and additional 20~25% are yet to be completely understood ( 17 ) .
crc develops over the course of more than 10 years , and entails multistep genetic events for tumor progression . a multistep genetic model of colorectal carcinogenesis
colorectal cancers occur as a result of oncogenes activation , along with the inactivation of tumor suppressor genes by mutations or loss of heterozygosity ( loh ) ( 18 ) .
mutations in at least four to five genes are required for the formation of a malignant tumor ( 18 ) .
the molecular pathogenesis of colorectal cancer is commonly classified into two main pathways of genomic instability : chromosomal instability ( cin ) and microsatellite instability ( msi ) pathways .
cin leads to an imbalance in chromosome number ( aneuploidy ) such as chromosome gains or losses ( including loh events at specific tumor suppressor gene loci ) , and is observed in 80~85% of sporadic crcs ( 18192021 ) . a mutation coupled with loh of the adenomatous polyposis coli ( apc ) tumor suppressor gene , followed by activating mutations of v - ki - ras2 kirsten rat sarcoma viral oncogene homolog ( kras ) , lead to the initial appearance of adenoma from normal colonic epithelium ( 18 ) .
apc mutations are typically observed in earliest stages of tumors , including in adenomas as small as 0.5 cm ( 22 ) .
this indicates that a functional loss of apc , which is part of the wnt signaling pathway , plays a role in early tumorigenesis .
in contrast , msi , which is observed in 15~20% of sporadic crcs ( 21 ) , is a hypermutable phenotype caused by impaired dna mismatch repair ( mmr ) system to correct errors such as single - base mismatches and insertion - deletion loops that spontaneously occur during dna replication ( 2324 ) .
msi arises from impaired dna mmr genes , such as mutl homolog 1 ( mlh ) 1 , muts homolog ( msh ) 2 , and to a lesser degree msh6 ( 2526 ) .
faulty dna fidelity caused by defects in dna mmr causes frameshift and point mutations , mainly in repetitive sequences ( microsatellites ) ( 26 ) .
lynch syndrome is a well - established hereditary predisposition to colorectal cancer caused by a germline mutation in a dna mmr gene .
however , 12% of msi exhibit sporadic crc with cin ( 27 ) . because pathways are not mutually exclusive , crc can display features of multiple pathways .
i colon cancer , surgery is the definitive treatment without the use of adjuvant chemotherapy .
benefit of adjuvant chemotherapy for patients with a stage ii disease is highly debatable due to minimal gains in overall response .
however , adjuvant chemotherapy is generally acceptable for the use patients with node - positive cancer following surgery ( high - risk stage ii disease ) ( 29 ) .
adjuvant chemotherapy is required for all patients with a stage iii colon cancer after surgical resection , which have a high risk of recurrence of 15~50% ( 30 ) .
conventional chemotherapeutic agents include 5-fluorouracil ( 5-fu ) with leucovorin ( lv ) , capecitabine ( an orally administered prodrug of 5-fu ) , oxaliplatin , and irinotecan .
adjuvant chemotherapy using the 5-fu / lv regimens following surgery provides significant reduction in mortality by 22% and improves event - free survival by 35% ( 31 ) . to improve disease - free survival ( dfs ) and overall survival ( os ) ,
currently , folfox ( 5-fu / lv and oxaliplatin ) or folfiri ( 5-fu / lv and irinotecan ) is widely used for standard first- or second - line treatment in patients with node - positive colon cancer following surgical resection ( 3233 ) .
however , folfox and folfiri demonstrated greater toxicities , including grade 3 or higher acute toxicity , compared to 5-fu / lv alone ( 34 ) . in 2004 , targeted therapeutic agents for metastatic colorectal cancer , such as anti - epidermal growth factor receptor ( egfr ) antibodies ( cetuximab and panitumumab ) , vascular endothelial growth factor ( vegf ) inhibitors ( bevacizumab , ziv - aflibercept , and ramucirumab ) , and a multikinase inhibitor ( regorafenib ) , were approved by the us food and drug administration ( fda ) for combinational use with standard chemotherapy treatment ( 35 ) . currently , there are various regimens of combinational therapies using targeted therapeutic agents and conventional chemotherapy drugs ( 36 ) .
treatment strategy is often dependent on tumor - specific factors , such as kras and/or v - raf murine sarcoma viral oncogene homolog b ( braf ) mutation , and condition of patients and their disease progression following first - line treatment ( 3336 ) .
since ras mutations lead to continuous activation of signaling pathways downstream of egfr , the use of anti - egfr antibodies is effective in patients with wild - type kras exon 2 , but not in patients with kras and/or braf mutations .
thus , approximately 30~40% of crcs are associated with kras gene mutation and do not respond to anti - egfr antibodies ( 37 ) . over the past decade ,
multidisciplinary advances in the treatment of metastatic colorectal cancer with targeted therapy have statistically improved progression - free and overall survivals . despite these advances , only a relatively small effect on survival outcomes was observed , especially in patients with distant metastatic disease .
dendritic cell ( dc ) therapy is one of several strategies in therapeutic cancer vaccines .
sipuleucel - t , a dc - based cancer vaccine , has been approved by the us fda for advanced prostate cancer in 2011 .
the goal of dc vaccination is to elicit anti - tumor response by inducing tumor - specific effector t cells .
dcs are generated ex vivo by culturing hematopoietic progenitor cells or monocytes with a combination of cytokines and then pulsing them with antigens ex vivo ( 38 ) .
there are various type of cancer antigens for dcs loading , including mutated and non - mutated antigens ( 39 ) .
now , we can find 9 clinical trials for the treatment of patients with crc from 2004 to 2015 ( table 1 ) .
antigens that are most commonly used for colorectal cancer include carcinoembryonic antigen ( cea ) peptides ( 4041 ) , melanoma - associated antigen ( mage ) from allogeneic melanoma cell lysates ( 4243 ) , and autologous cell lysates from biopsy material ( 44 ) .
while cea expression in normal colon epithelial cells is relatively low , it is over expressed in most colorectal carcinomas as well as in many cancers ( 45 ) .
therefore , cea has been the major immunological target of dc - based cancer vaccines for colorectal cancer .
clinical trials of cea - pulsed dcs demonstrated its immune - stimulatory capacity and it was well tolerated in patients without any observable toxicities .
however , the overall clinical response was rather unimpressive ( 404146 ) , which may reflect severely impaired immune functions in patients with excessive tumor burdens and tumor immunoediting mechanisms . nevertheless , several clinical studies of mage - pulsed dcs showed 24~40% clinical benefit rate with durable responses and tumor regression ( 4243 ) . moreover , regulatory t cell levels declined upon dc vaccination ( 42 ) .
recently , hunyadi j. and colleagues demonstrated that autologous tumor cell lysates - loaded dcs led to an increase in 6-years survival rate in colorectal cancer patients and more efficient induction of t - lymphocytes proliferation in vitro when compared to cea - pulsed dcs ( 44 ) .
however , because of limited number of patients , additional evaluations in large - scale clinical trials are needed .
although significant advances have been made over the past decade , further studies are required to fully determine the potential antitumor effects of dc vaccination for colorectal cancer .
adoptive cell therapy ( act ) for metastatic melanoma was first described in 1988 ( 47 ) . in act , tumor - infiltrating lymphocytes ( tils ) are collected from solid tumor specimen , and are activated and expanded ex vivo .
subsequently , tils are administered intravenously to the autologous patient . in 2002 , dudley me et al
. demonstrated that host immunodepletion using cyclophosphamide and fludarabine prior to til infusion resulted in dramatic improvement in clinical outcomes for patients with metastatic melanoma ( 48 ) .
although act has been evaluated in various cancers , only a small number of act trials for crc have been performed . in a phase i clinical trial ,
14 patients with colorectal liver metastases received act with tils in combination with high - dose il-2 ( 49 ) .
unfortunately , no significant difference in dfs rates was observed between tils therapy and conventional chemotherapy .
a novel act approach for crc was conducted in two separate studies ; sentinel lymph node ( sln ) t lymphocytes were used in a pilot study ( seven with stage ii - iii and nine with stage iv crc ) ( 50 ) and in a phase i / ii study ( 46 with stage i - iii and nine with stage iv crc ) ( 51 ) , respectively .
all patients with stage iv disease responded to the treatment with the following outcomes : four stable disease , one partial response , and four complete response with no detectable cancer cells remaining .
the overall survival of act - treated patients was also significantly improved to 2.6 years compared to 0.8 years in conventionally - treated control patients .
the latest phase i / ii study also showed favorable clinical outcomes ( 51 ) .
the median overall survival of sln - t lymphocytes - treated and control groups in stage iv patients were 28 and 14 months , respectively .
in the two studies , no significant toxicity was observed following sln - t lymphocytes treatment .
these studies demonstrated that sln - t lymphocyte immunotherapy is indeed feasible and safe for patients with metastatic crc .
however , surgeon 's proficiency may be required for the intraoperative sentinel node detection . in a phase i study of autologous genetically engineered t cells ,
all patients developed severe colitis despite showing a decrease in serum cea levels by cea - specific t cells ( 52 ) .
cytokine - induced killer ( cik ) cells are ex vivo - expanded lymphocytes used for cancer immunotherapy .
cik cells are generated by culturing peripheral blood mononuclear cells ( pbmcs ) with a combination of il-2 and anti - cd3 monoclonal antibodies for 14 days ( 53 ) .
cik cells consist of heterogeneous cell population , mainly cd3cd56 , cd3cd56 , and a minor population of cd3cd56 cells .
importantly , cd3cd56 cells are the distinguishing cell population among the cik cells and have the most potent cytotoxic function ( 53 ) .
cd3cd56 cells originated from cd3cd8cd56 cells but not from cd3cd56 cells during the ex vivo expansion ( 54 ) .
cik cell cytotoxicity is mediated by perforin release and dependent on several activating receptors such as nkg2d , nkp30 , and dnam-1 ( 5556 ) .
cik cells also exhibit non - specific and non - mhc - restricted cytotoxicity ( 56 ) . over the past decade
, cik cell therapy has been evaluated in numerous clinical studies in patients with various types of cancer , such as hepatocellular carcinoma , non - small cell lung cancer , renal cell carcinoma , and gastric cancer ( 57 ) .
cik cell therapy can be used as a postoperative adjuvant treatment as well as a palliative treatment following standard therapies .
cik cell therapy was evaluated in a limited number of clinical studies in patients with colorectal cancer . in a retrospective study , 21 of 96 colorectal cancer patients who underwent surgery as well as adjuvant chemotherapy received one to three cycles of cik cell transfusion for immunotherapy ( 58 ) .
patients in the cik - treated group had significant improvement in their 2-year dfs rates than those in the control group ( 59.6524.80% vs 29.356.39% ) .
other studies used cik in a combination therapy with dcs , specifically tumor lysate - pulsed dcs . in 2014 , two clinical studies of dc vaccine and cik cell combinational therapy for colorectal cancer patients were published .
one study demonstrated that overall survival rates were significantly improved in the dc - cik therapy compared to the control group ( p=0.04 ) ( 59 ) .
moreover , most patients that received dc - cik therapy displayed improvement in quality of life , including physical strength , appetite , sleep , and body weight .
gao , d. and colleagues evaluated the clinical benefits of dc - cik therapy in 54 gastric and colorectal patients following surgery with or without chemo - radiotherapy ( 60 ) .
the study demonstrated that dfs and 5-year survival of colorectal cancer patients were significantly prolonged in dc - cik treatment groups ( dfs rate : 66% and 5-year survival rates : 75% ) when compared to patients in control groups ( dfs rate : 8% and 5-year survival rates : 15% ; p<0.01 ) .
il-12 levels were significantly increased in patients that received the dc - cik treatment . besides fever ,
since they did not compare the efficacy of combination therapy with each alone , we could not clarify whether combination therapy is better than each therapy .
however , when we considered the efficacy of the single therapy of dcs and cik cells described above , we could presume that the combination of cik and dc might exert better antitumor activity than each alone . here
, we provide additional evidence that cik cell therapy can effectively prevent growth of colorectal cancers in a xenograft mouse model .
hypaque density gradient centrifugation , washed three times with pbs , and cultured in the presence of immobilized anti - cd3 antibody ( 5 g / ml ) and recombinant human il-2 ( 700 u / ml ) for five days .
the cell suspension was further incubated in complete medium containing il-2 only ( 170 u / ml ) for nine days .
thereafter , medium containing il-2 was replenished every 2 or 3 days . on day 14
cik cells comprised of 94% cd3 , 3% cd3cd56 , 16% cd3cd56 , 14% cd4 , and 81% cd8 cells ( fig .
1a ) . to examine the cytotoxicity of cik cells , two cell lines , sw620 and k-562 were used .
sw620 cells are metastatic human colon cancer cell lines derived from lymph node ( 61 )
. k-562 cell lines are human leukemic cells commonly used as reference target cells of nk cells due to the very low level of mhc class i antigens on their cell membrane ( 62 ) .
a 4-h cr release assay revealed that cik cells were able to kill 14% , 22% , and 36% of sw620 cells at effector : target ratios of 10:1 , 30:1 , and 100:1 , respectively ( fig .
moreover , cik cells showed strong cytotoxicity against k562 cells , a known nk - sensitive target .
a nude mouse xenograft assay was used to examine the in vivo antitumor activity of cik cells .
sw620 cells ( 610 ) in 300 l of pbs were injected subcutaneously on day 0 , followed by intravenous once - a - week injection of cik cells and adriamycin ( adr ) . on day 25 , mice were sacrificed and the tumor mass and body weight ( to determine toxicity ) were measured . in control mice , sw620 cells grew to a tumor volume of 473190 mm ( n=7 ) ( fig .
cik cells injected at doses of 110 , 310 , and 1010 cells per mouse were able to inhibit tumor growth by 7% , 53% , and 73% , respectively .
cik cells injected at doses of 110 , 310 and 1010 cells per mouse reduced tumor weight by 10% , 54% and 73% , respectively ( fig .
thus , our preclinical efficacy data showed that cik cells were able to kill sw620 cells in vitro and in vivo , suggesting that cik cells might be a good immunotherapy candidate for colorectal cancer .
patients with metastatic colorectal cancer have significant risk of recurrence following surgery and conventional adjuvant therapy . although advances in therapeutic agents for crc have reduced the risk of recurrence and increased overall survival , patients with metastatic disease have a poor prognosis and a particularly low 5-year survival rates of less than 10% .
additionally , current combination therapy is occasionally discontinued due to a grade 3~4 toxicity with negative effects on quality of life ( 63 ) .
overall quality of life should be considered for patients with stage iv disease for palliative therapy .
also , elderly patients with stage iii disease often avoid adjuvant chemotherapy due to anxiety of old age , comorbidities , side effects , and perceived minimal benefit ( 64 ) .
clinical and preclinical studies of crc treatment with dcs , t cells , and cik cells showed promising outcomes , although only limited information have been available till recently . among the immune effector cells , cik cells have certain advantages in a clinical application : they are relatively easy to generate and expanded in large - scale from pbmcs , their non - mhc - restricted cytotoxic activity could eliminate mhc class i - negative tumor cells , and importantly , cik cell therapy causes mild , transient , and easy - to - manage side effects . over the last decades , cik cell therapy has been evaluated in numerous clinical studies in patients with various cancers . in most patients ,
combination therapy using conventional agents and cik cells showed superior clinical outcomes than standard therapy alone .
clinical studies previously reported by others and our preclinical data suggest that immunotherapy of crc with cik cells can be a promising strategy to limit the growth and metastasis of crc .
 colorectal cancer is the third leading cancer worldwide . 
 although incidence and mortality of colorectal cancer are gradually decreasing in the us , patients with metastatic colorectal cancer have poor prognosis with an estimated 5-year survival rate of less than 10% . over the past decade , advances in combination chemotherapy regimens for colorectal cancer have led to significant improvement in progression - free and overall survival . 
 however , patients with metastatic disease gain little clinical benefit from conventional therapy , which is associated with grade 3~4 toxicity with negative effects on quality of life . in previous clinical studies , 
 cell - based immunotherapy using dendritic cell vaccines and sentinel lymph node t cell therapy showed promising therapeutic results for metastatic colorectal cancer . in our preclinical and previous clinical studies , 
 cytokine - induced killer ( cik ) cells treatment for colorectal cancer showed favorable responses without toxicities . here , we review current treatment options for colorectal cancer and summarize available clinical studies utilizing cell - based immunotherapy . based on these studies , 
 we recommend the use cik cell therapy as a promising therapeutic strategy for patients with metastatic colorectal cancer . 
INTRODUCTION
THE MOLECULAR PATHOGENESIS OF COLORECTAL CANCER
CURRENT TREATMENT OF COLORECTAL CANCER
DENDRITIC CELL THERAPY FOR COLORECTAL CANCER
T CELL THERAPY OF COLORECTAL CANCER
CYTOKINE-INDUCED KILL CELL THERAPY FOR COLORECTAL CANCER
CONCLUSION