Skip to main content
NCBI home page
Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2015 Apr 10;141(10):1827–1833. doi: 10.1007/s00432-015-1968-4

Prospective study of the safety and efficacy of a pancreatic cancer stem cell vaccine

Mao Lin 1, Yuan-Ying Yuan 1, Shu-Peng Liu 1, Juan-Juan Shi 1, Xin-An Long 1, Li-Zhi Niu 1, Ji-Bing Chen 1,✉, Qiao Li 2,✉, Ke-Cheng Xu 1
PMCID: PMC11823924  PMID: 25860618

Abstract

Introduction

In this trial, we isolated and cultured pancreatic cancer stem cells (CSCs) to produce a vaccine and prospectively evaluated its safety and efficacy in low-, medium-, and high-dose groups.

Material and methods

Between February and October 2014, we enrolled 90 patients who met the enrollment criteria and assigned them to three groups (n = 30). CSC-specific and CSC-non-specific immunity pre- and post-vaccination were compared by Dunnett’s multiple comparison test (one-way ANOVA). The data are presented as the mean±standard deviation. Local and systemic adverse events were recorded in the nursing records and compared using the Chi-square test. All statistical analyses were conducted using GraphPad software (GraphPad, San Diego, CA, USA).

Results

Throughout the trial, an injection site reaction was the most common reaction (54 %), and fever was least common (9 %). The incidence of these side effects did not vary among the three groups. When the pre- and post-vaccination immunity was compared, we found that both CSC-nonspecific and CSC-specific responses were significantly increased in the high-dose group.

Conclusion

This study is the first clinical trial of a pancreatic CSC vaccine and preliminarily proves its safety and efficacy.

Keywords: Cancer stem cell, Vaccine, Pancreatic cancer, Safety, Efficacy

Introduction

Pancreatic cancer-related mortality is almost invariably due to metastasis (Siegel et al. 2011), which occurs in more than half of the patients diagnosed with this disease, often years after the primary tumor is diagnosed and removed (Jemal et al. 2010). The therapeutic alternatives for metastatic pancreatic cancer are mainly involve the systemic administration of cytotoxic chemotherapeutic agents (Heinemann et al. 2008; Wolff 2007), and the long-term survival is greatly dependent on the nature of the metastases and physical condition of patients (Moore et al. 2007; Tokh et al. 2012). Many pilot studies have confirmed that cryoablation, primarily in early and late stages of pancreatic cancer, is associated with improved therapeutic efficacy and fewer side effects (Xu et al. 2008a, b). Indeed, cryotherapy or cryoimmunotherapy may prolong the survival of patients with metastasis (Niu et al. 2013), and immunotherapy may have powerful therapeutic effects under low-burden load conditions (Kaneko et al. 2005; Koido et al. 2011).

Traditional DC-CIK immunotherapy supplements the lymphocyte count and function effectively, but has no tumor specificity (Marten and Buchler 2008; Thanendrarajan et al. 2011) and results in poor memory immune responses, which are important for the long-term prevention of tumor recurrence. Currently, accumulating evidence suggests that a tumor cell subpopulation with distinct stem cell-like properties (the so-called cancer stem cells, CSCs) is responsible for tumor initiation, invasion, and metastasis in patients with pancreatic cancer (Habib and Saif 2013; Kumar et al. 2013; Xu 2013). In the laboratory, CSCs are identified by the CD44 marker and their ability to form new pancreatic cancer colonies through serial transplantations in immunodeficient hosts, thus reestablishing tumor heterogeneity (Ohara et al. 2013; Palagani et al. 2012; Wood 2014). Furthermore, while pancreatic cancer stem cells can also be identified by other surface markers, the CD44 MicroBead kit is the only commercialized kit that has been validated for pancreatic cancer stem cell identification.

To assess the feasibility of generating CSC vaccines for clinical use, we harvested peripheral blood and tumor specimens from patients with pancreatic adenocarcinoma. Lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs), while CSCs were isolated from the tumor specimens. Cytotoxic T cell- and antibody-mediated CSC-specific and CSC-nonspecific immunity before and after the vaccination, as well as adverse event occurrence were compared.

Materials and methods

Ethics

This clinical trial was registered in the US National Institutes of Health (ID: NCT02074046; Ph1/Ph2) and approved by the Ethics Committee of Guangzhou Fuda Cancer Hospital. In accordance with the Declaration of Helsinki, written informed consent was obtained from each participant in Fuda Cancer Hospital.

Patient selection

This was a prospective study of the therapeutic effects of a CSC vaccine for pancreatic adenocarcinoma patients enrolled between February and October 2014. We enrolled 90 patients who fulfilled the following criteria: (1) had at least one resectable tumor, and other tumors could be well controlled by cryosurgery, brachytherapy, or chemotherapy, (2) the expected survival was >3 months, (3) were between 30 and 75 years of age, (4) the Karnofsky performance status score was >70, and (5) immune parameters fell with the following normal ranges: total T cells, 603–2990/μL; cytotoxic T cells, 125–1312/μL; helper T (Th) cells, 441–2156/μL; platelets ≥80 × 109/L; white blood cells ≥3 × 109/L; neutrophils ≥2 × 109/L; hemoglobin ≥90 g/L; prothrombin time international normalized ratio, 0.8–1.5; adequate hepatic function (bilirubin <20 μM, aminotransferase <60 U/L); and renal function (serum creatinine <130 μM, serum urea <10 mM), (6) absence of level 3 hypertension, severe coronary disease, myelosuppression, respiratory disease, acute or chronic infection, and autoimmune diseases. The contraindications for participation were T cell lymphoma, ongoing organ transplant, or patients presenting within 7 days of systemic chemotherapy. The enrolled patients were allocated to three groups (n = 30) with parallel assignments.

Preparation of CSC vaccine

The CSC vaccine contained three components: pancreatic CSC fragments, a multioil fat emulsion (C6–24; Fresenius Kabi GmbH, Graz-Puntigam, Austria), and mannatide (Duokang; Chengdu Lier Pharmaceutical, Chengdu, China). The total vaccine volume was 0.5 mL: 0.3 mL CSC/saline solution (105, 3 × 105, and 5 × 105 CSCs for the low-, medium-, and high-dose groups, respectively), and 0.1 mL of each of the remaining components.

The CSC preparation process involved seven steps: (1) solid tumor resection (e.g., primary lesions or metastases in pancreas, liver, or lung with a diameter usually >4 cm), (2) preparation of single-cell suspensions using a human Tumor Dissociation Kit (no. 130-095-929; Miltenyi Biotec, Bergisch Gladbach, Germany), (3) CSC separation using a human CD44 MicroBead Kit (no. 130-095-194; clone MC56; Miltenyi Biotec), (4) CSC amplification and culture using a human StemMACS MSC Expansion Media Kit XF (no. 130-104-182; Miltenyi Biotec), (5) detection of cell purity by phycoerythrin (PE) antihuman CD44s (no. 130-095-180; clone DB105; Miltenyi Biotec), (6) cell counting and dilution to different concentrations, and (7) CSC lysis by repeated freezing and thawing five times. All steps were performed in accordance with the corresponding manufacturer instructions.

Vaccinations and tests

We used a vaccination schedule that has been previously established in an animal study from our group (Ning et al. 2012); the vaccination involved two subcutaneous injections on either side of the deltoid muscle, interspersed by a 1-week interval. Peripheral blood (~30 mL) was drawn for in vitro testing before and 1 week after vaccination (Fig. 1).

Fig. 1.

Fig. 1

Open in a new tab

Vaccination and test schedule, and trend chart of CSC numbers. Broken lines represent the gradual amplification of CSCs and single consumption during vaccination or testing

Peripheral blood (2 mL) was used for detecting CSC-nonspecific immunity by flow cytometry (FACSCanto™ II; BD, Franklin Lakes, NJ, USA). Multitest 6-color TBNK Reagent (no. 644611; BD) was used to detect the number of lymphocyte subsets; Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II (no. 551809; BD) was used to detect cytokine expression levels. All steps were performed in accordance with the corresponding manufacturer instructions.

Using Ficoll-Hypaque density centrifugation, PBMCs and plasma were harvested from the remaining blood samples, and cytotoxic T cell- and antibody-mediated CSC-specific immunity before and after vaccination were compared. CSC-specific testing included CSC binding by immune plasma, and complement-dependent cytotoxicity (CDC) and cytotoxic T lymphocyte (CTL) tests. Plasma CSC-binding antibodies were detected using fluorescein isothiocyanate antihuman immunoglobulin G (IgG, no. 130-099-229; clone IS11-3B2.2.3; Miltenyi Biotec) using previously described protocols (Ning et al. 2012). T lymphocytes were separated by human CD3 MicroBeads (no. 130-050-101; Miltenyi Biotec), activated by antihuman CD3/28 (no. 300314/302934, BioLegend, San Diego, CA, USA) and expanded using interleukin (IL)-2 (no. 200-02, PeproTech, Rocky Hill, NJ, USA), while DCs were induced and cultured using IL-4 (no. 200-04, PeproTech) and granulocyte–macrophage colony-stimulating factor (no. 300-03, PeproTech), as previously described (Niu et al. 2013).

Evaluation and statistical analysis

Complications were recorded and classified in accordance with the Common Terminology Criteria of Adverse Events version 4.02. Radiographic local tumor control was assessed using image-guided tumor ablation criteria (Goldberg et al. 2005). Patient demographics were compared among the three groups before vaccination by two-way analysis of variance (ANOVA); CSC-specific and CSC-nonspecific immunity pre- and post-vaccination were compared by Dunnett’s multiple comparison test (one-way ANOVA), and data are presented as the mean ± standard deviation. Local and systemic adverse events were marked in the nursing records and compared using the Chi-square test. All analyses were conducted using GraphPad software (GraphPad, San Diego, CA, USA).

Results

Identification of CSCs

As per the protocol depicted in Fig. 1, tumors were surgically resected from the patients, and CSCs were then separated in the lab. Before separation, CSC purity was 3 ± 1.1 %; after separation, CSC purity increased up to 93 ± 12 % (Fig. 2). All CSCs separated from the tumor mass were sufficient for culture, vaccination, and testing, and CD44+ cell purity after 2- and 4-week culture was similar to that immediately following separation (data not shown).

Fig. 2.

Fig. 2

Open in a new tab

CSCs in pancreatic cancer mass. a CSC purity in tumor cell suspension before separation. b CSC purity in separation products obtained using the human CD44 MicroBead Kit

Clinical data

The patients (n = 90) enrolled in this study were from China (36/90), Indonesia (25/90), and Malaysia (29/90). Analysis of the pre-treatment data revealed no statistical differences in patient demographics among the three groups (Table 1). Immune monitoring was carried out both pre-vaccination (Test 1) and post-vaccination (Test 2) (Table 2; Fig. 4).

Table 1.

Patient demographics

Patient characteristics before vaccination Total (n = 90) Low-dose group (n = 30) Medium-dose group (n = 30) High-dose group (n = 30) P value
Gender (male/female) 43/47 16/14 15/15 12/18 >0.05
Median age (years) 55 58 54 47 >0.05
Differentiated degree of adenocarcinoma
 High 44 16 15 13 >0.05
 Moderate 34 10 12 12 >0.05
 Poor 12 4 3 5 >0.05
Clinical stage
 II 30 11 7 12 >0.05
 III 36 12 14 10 >0.05
 IV 24 7 9 8 >0.05
Karnofsky performance status
 80 26 7 9 10 >0.05
 90 31 11 11 9 >0.05
 100 33 12 10 11 >0.05
Chemotherapy 48 16 17 15 >0.05
Cryosurgery 51 14 18 19 >0.05
Surgery 90 30 30 30 >0.05
Open in a new tab

Factors of low-, middle-, and high-dose group were compared by two-way ANOVA

Table 2.

Comparison of lymphocyte number and function before and after vaccination

Test items of lymphocyte Testing results
Test 1 (n = 90) Test 2 (low-dose group) Test 2 (medium-dose group) Test 2 (high-dose group)
Number (cell/μL)
Total T cell 1392 ± 47 1410 ± 62 1467 ± 74 1790 ± 79**
CD8+T cell 504 ± 12 566 ± 13 606 ± 17* 650 ± 15*
CD4+T cell 64 ± 16 681 ± 28 704 ± 21 739 ± 18*
NK cell 293 ± 17 407 ± 37 437 ± 49 510 ± 58**
B cell 192 ± 14 245 ± 12 348 ± 22 434 ± 30**
Function (pg/mL)
IL-2 10 ± 3 12 ± 4* 16 ± 3* 21 ± 4**
TNF-β 3 ± 2.4 4 ± 3.5 8 ± 2 16 ± 2.8*
IFN-γ 5 ± 3.1 6 ± 2.9 9 ± 3 15 ± 3.4*
IL-4 12 ± 2 11 ± 2.7 9 ± 3.2 10 ± 3
IL-6 18 ± 3.2 20 ± 3.1 18 ± 4 21 ± 9.2
IL-10 11 ± 2.6 12 ± 3.3 11 ± 2.5 10 ± 3.3
Open in a new tab

Every cell subset or cytokine were analyzed using Dunnett’s multiple comparison test (one-way ANOVA)

NK cell natural killer cell, IL interleukin, TNF tumor necrosis factor, IFN interferon

* P < 0.05; ** P < 0.01; *** P < 0.001

Fig. 4.

Fig. 4

Open in a new tab

Comparison of CSC-specific immunity pre- and post-vaccination. a Flow cytometry results of CSC-specific antibody levels in immune plasma and IgG levels. b CSC targeting by CSC-primed CDC; 105 viable CSCs were incubated with immune plasma for 1 h (control group: no immune plasma), then rabbit complement was added and incubated for another 1 h. Viable cells were then counted under a microscope after trypan blue staining. c CSC targeting by CSC-primed CTLs; CSC killing was evaluated using a lactate dehydrogenase release assay. A higher percentage of cytotoxicity indicated more cell lysis. *P < 0.05

Adverse events

Throughout the trial, the adverse events experienced by the patients were recorded and included local (mainly injection site reaction or localized edema) and systemic (mainly chills, fatigue, or fever) reactions. The most common reaction was an injection site reaction (54 %), while fever was the least common (9 %). Other possible side effects such as blood or bone marrow changes were not detected. All adverse events were Grade 1, and all reactions were resolved within 1 day of treatment of the specific symptoms and did not recur. Adverse event occurrence was compared using the Chi-square test; there was no difference among the three groups (P = 0.996, Fig. 3).

Fig. 3.

Fig. 3

Open in a new tab

Local and systemic adverse events. The incidences of each Grade 1 adverse event that occurred in the three groups are depicted in separate columns; figures above the columns indicate the average incidences in all patients

CSC-nonspecific and CSC-specific immunity

Lymphocyte count and function before (Test 1) and after (Test 2) vaccination were compared, with patients in all three groups showing an increase in these parameters after vaccination (Table 2). Patient pre-vaccination data were merged and compared with that obtained post-vaccination (Table 2); the cell numbers for all subsets examined were significantly higher after vaccination in the high-dose group. Additionally, Th1-type cytokine levels were increased in the medium- and high-dose groups, while Th2-type cytokine levels were essentially unchanged.

CSC-specific immunity was also compared (Fig. 4) by examining CSC binding by immune plasma: The frequency of IgG-positive cells was significantly higher in all the groups post-vaccination (P < 0.001, Fig. 4a);

CSC lysis by immune plasma, as determined by the percentage of viable cells after CDC (91 ± 10 %; Test 1), was increased in the high-dose group (55 ± 19 %, P < 0.01; Fig. 4b). When CSC destruction by CTLs was examined, the cytotoxicity effects (for three effect/target proportions) were all significantly increased in the high-dose group (P < 0.01, Fig. 4c).

Discussion

The subject of some controversy, CSCs are considered capable of symmetric or asymmetric self-renewal, are resistant to standard chemotherapies or radiotherapies, and are less differentiated and tumorigenic, as evidenced by their capacity to establish tumors in immunodeficient mice even when a low number of cells is injected (Dick 2008; Visvader 2011). As the failure of current therapies to control cancer can be attributed to their inability to eliminate CSCs, it is critical to develop strategies that eliminate these stem cell-like tumor cells. Recent data suggest that immune-based approaches may be particularly attractive prospects for targeting CSCs: One strategy is to target CSCs via monoclonal antibodies such as CD47 (Majeti et al. 2009) and CD123 (Jin et al. 2009), which have been shown to eradicate leukemia stem cells in preclinical models. Another approach, which has also produced good effects in animal models, involves harnessing cellular immune components, such as antitumor T cells (Sato et al. 2009; Schatton and Frank 2009) or antitumor DCs (Ning et al. 2012; Xu et al. 2009), Currently, commercial CSC separation and amplification kits are widely used, offering technical guarantees for the clinical application of this vaccine. This study represents the first trial of a pancreatic CSC vaccine in clinical use and is a step forward in the exploration of cellular immune responses targeting CSCs. Importantly, no blood or bone marrow changes were detected in any patient, and local or systemic side effects that appeared could be alleviated within the day. From the perspective of efficacy, CSC-specific and CSC-nonspecific lymphocytes were both significantly increased as the number of CSCs in the vaccine was increased. Our findings support that the vaccination resulted in an activation of Th1 immune pathways (as evidenced by increases in IL-2, TNF-α, and IFN-γ levels post-vaccination) and increased the number of NK cells and T cells.

Additionally, our protocol was tailored to maximize the safety and efficacy of the vaccine: (1) all patients underwent cytoreductive surgery, radiotherapy, or cryosurgery to reduce the tumor burden and then received the vaccination. The organ-like structural environment of tumors is very hostile toward immune cells and hence may play a role in local immune tolerance (Plate 2011); a low tumor burden provides the necessary conditions for the in vivo survival and amplification of specific lymphocytes. (2) DC activation plays a central role in pancreatic cancer, where the level of tumor-specific lymphocytes decreases prior to tumor recurrence and increases again with immunization (Kaneko et al. 2005; Koido et al. 2011). Our experience with animal experiments established the foundations of how and when to vaccinate (Ning et al. 2012); although the drawing of extra blood for DC preparation is difficult, the addition of adjuvants (Mannatide Injection) compensated for this drawback and produced a good immune effect. (3) Ongoing CSC cultures ensured antigen activity during vaccination and detection. (4) Given the differences between allogeneic CSCs from different individuals, our approach of generating patient-derived CSC vaccines for each study subject guaranteed a CSC-specific response and appropriate activation of the T cell repertoire.

In summary, this study provides novel and preliminary evidence of the safety and efficacy of the pancreatic CSC vaccine in a clinical trial, but the vaccine requires further refining. At the cellular level, CSC vaccination may modulate immune responses via several mechanisms. For example, CSCs may selectively not express tumor-associated antigens associated with differentiation and may therefore be resistant to immune-mediated rejection or evade host immunosurveillance through the absence or downregulation of major histocompatibility complex class I molecules (Aptsiauri et al. 2007). Therefore, the mechanism by which the pancreatic CSC vaccine strengthens the specific and nonspecific response requires further investigation. From the viewpoint of a long-term curative effect, the benefit of this vaccine on progression-free survival and overall survival also requires further research.

Acknowledgments

We would like to thank the native English speaking scientists of Elixigen Company for editing our manuscript. This work was supported by the Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences (Program No. 2008DP173344).

Conflict of interest

We declared that we have no conflict of interest.

Contributor Information

Ji-Bing Chen, Email: jibingchen398@163.com.

Qiao Li, Email: qiaoli@umich.edu.

References

  1. Aptsiauri N, Cabrera T, Mendez R, Garcia-Lora A, Ruiz-Cabello F, Garrido F (2007) Role of altered expression of HLA class I molecules in cancer progression. Adv Exp Med Biol 601:123–131 [DOI] [PubMed] [Google Scholar]
  2. Dick JE (2008) Stem cell concepts renew cancer research. Blood 112:4793–4807. doi:10.1182/blood-2008-08-077941 [DOI] [PubMed] [Google Scholar]
  3. Goldberg SN, Grassi CJ, Cardella JF, Charboneau JW, Dodd GD III, Dupuy DE et al (2005) Image-guided tumor ablation: standardization of terminology and reporting criteria. J Vasc Interv Radiol 16:765–778. doi:10.1097/01.RVI.0000170858.46668.65 [DOI] [PubMed] [Google Scholar]
  4. Habib M, Saif MW (2013) Pancreatic cancer stem cells: their role in pancreatic cancer patient outcomes and what is future? JOP 14:401–404 [DOI] [PubMed] [Google Scholar]
  5. Heinemann V, Boeck S, Hinke A, Labianca R, Louvet C (2008) Meta-analysis of randomized trials: evaluation of benefit from gemcitabine-based combination chemotherapy applied in advanced pancreatic cancer. BMC Cancer 8:82. doi:10.1186/1471-2407-8-82 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin 60:277–300. doi:10.3322/caac.20073 [DOI] [PubMed] [Google Scholar]
  7. Jin L, Lee EM, Ramshaw HS, Busfield SJ, Peoppl AG, Wilkinson L et al (2009) Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cell 5:31–42. doi:10.1016/j.stem.2009.04.018 [DOI] [PubMed] [Google Scholar]
  8. Kaneko T, Goto S, Kato A, Akeyama A, Tomonaga M, Fujimoto K et al (2005) Efficacy of immuno-cell therapy in patients with advanced pancreatic cancer. Anticancer Res 25:3709–3714 [PubMed] [Google Scholar]
  9. Koido S, Homma S, Takahara A, Namiki Y, Tsukinaga S, Mitobe J et al (2011) Current immunotherapeutic approaches in pancreatic cancer. Clin Dev Immunol 2011:267539. doi:10.1155/2011/267539 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kumar R, Dholakia A, Rasheed Z (2013) Stem cell-directed therapies in pancreatic cancer. Curr Probl Cancer 37:280–286. doi:10.1016/j.currproblcancer.2013.10.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Majeti R, Chao MP, Alizadeh AA, Pang WW, Jaiswal S, Gibbs KD Jr et al (2009) CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell 138:286–299. doi:10.1016/j.cell.2009.05.045 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Marten A, Buchler MW (2008) Immunotherapy of pancreatic carcinoma. Curr Opin Investig Drugs 9:565–569 [PubMed] [Google Scholar]
  13. Moore MJ, Goldstein D, Hamm J, Figer A, Hecht JR, Gallinger S et al (2007) Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 25:1960–1966. doi:10.1200/JCO.2006.07.9525 [DOI] [PubMed] [Google Scholar]
  14. Ning N, Pan Q, Zheng F, Teitz-Tennenbaum S, Egenti M, Yet J et al (2012) Cancer stem cell vaccination confers significant antitumor immunity. Cancer Res 72:1853–1864. doi:10.1158/0008-5472.CAN-11-1400 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Niu L, Chen J, He L, Liao M, Yuan Y, Zeng J et al (2013) Combination treatment with comprehensive cryoablation and immunotherapy in metastatic pancreatic cancer. Pancreas 42:1143–1149. doi:10.1097/MPA.0b013e3182965dde [DOI] [PubMed] [Google Scholar]
  16. Ohara Y, Oda T, Sugano M, Hashimoto S, Enomoto T, Yamada K et al (2013) Histological and prognostic importance of CD44(+)/CD24(+)/EpCAM(+) expression in clinical pancreatic cancer. Cancer Sci 104:1127–1134. doi:10.1111/cas.12198 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Palagani V, El Khatib M, Kossatz U, Bozko P, Muller MR, Manns MP et al (2012) Epithelial mesenchymal transition and pancreatic tumor initiating CD44+/EpCAM+ cells are inhibited by gamma-secretase inhibitor IX. PLoS One 7:e46514. doi:10.1371/journal.pone.0046514 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Plate J (2011) Clinical trials of vaccines for immunotherapy in pancreatic cancer. Expert Rev Vaccin 10:825–836. doi:10.1586/erv.11.77 [DOI] [PubMed] [Google Scholar]
  19. Sato N, Hirohashi Y, Tsukahara T, Kikuchi T, Sahara H, Kamiguchi K et al (2009) Molecular pathological approaches to human tumor immunology. Pathol Int 59:205–217. doi:10.1111/j.1440-1827.2009.02353.x [DOI] [PubMed] [Google Scholar]
  20. Schatton T, Frank MH (2009) Antitumor immunity and cancer stem cells. Ann NY Acad Sci 1176:154–169. doi:10.1111/j.1749-6632.2009.04568.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Siegel R, Ward E, Brawley O, Jemal A (2011) Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin 61:212–236. doi:10.3322/caac.20121 [DOI] [PubMed] [Google Scholar]
  22. Thanendrarajan S, Nowak M, Abken H, Schmidt-Wolf IG (2011) Combining cytokine-induced killer cells with vaccination in cancer immunotherapy: more than one plus one? Leuk Res 35:1136–1142. doi:10.1016/j.leukres.2011.05.005 [DOI] [PubMed] [Google Scholar]
  23. Tokh M, Bathini V, Saif MW (2012) First-line treatment of metastatic pancreatic cancer. JOP 13:159–162 [PubMed] [Google Scholar]
  24. Visvader JE (2011) Cells of origin in cancer. Nature 469:314–322. doi:10.1038/nature09781 [DOI] [PubMed] [Google Scholar]
  25. Wolff RA (2007) Chemotherapy for pancreatic cancer: from metastatic disease to adjuvant therapy. Cancer J 13:175–184. doi:10.1097/PPO.0b013e318074e6c3 [DOI] [PubMed] [Google Scholar]
  26. Wood NJ (2014) Pancreatic cancer: pancreatic tumour formation and recurrence after radiotherapy are blocked by targeting CD44. Nat Rev Gastroenterol Hepatol 11:73. doi:10.1038/nrgastro.2014.1 [DOI] [PubMed] [Google Scholar]
  27. Xu L (2013) Cancer stem cell in the progression and therapy of pancreatic cancer. Front Biosci (Landmark Ed) 18:795–802 [DOI] [PubMed] [Google Scholar]
  28. Xu KC, Niu LZ, Hu YZ, He WB, He YS, Li YF et al (2008a) A pilot study on combination of cryosurgery and (125)iodine seed implantation for treatment of locally advanced pancreatic cancer. World J Gastroenterol 14:1603–1611. doi:10.3748/wjg.14.1603 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Xu KC, Niu LZ, Hu YZ, He WB, He YS, Zuo JS (2008b) Cryosurgery with combination of (125)iodine seed implantation for the treatment of locally advanced pancreatic cancer. J Dig Dis 9:32–40. doi:10.1111/j.1443-9573.2007.00322.x [DOI] [PubMed] [Google Scholar]
  30. Xu Q, Liu G, Yuan X, Xu M, Wang H, Ji J et al (2009) Antigen-specific T-cell response from dendritic cell vaccination using cancer stem-like cell-associated antigens. Stem Cells 27:1734–1740. doi:10.1002/stem.102 [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Cancer Research and Clinical Oncology are provided here courtesy of Springer

RESOURCES