Introduction

Colorectal carcinoma (CRC) is the third most common malignancy and the second most common cause of cancer-related death. Its incidence rate has fallen from 60.5 per 100,000 people in 1976 to 46.4 in 2005, and more dropped to 38.7 in 2016. However, the proportion of rectal cancer within CRC has increased from 27% in 1995 to 31% in 2019.1 First-line treatment for metastatic rectal cancer is 5-FU-based cytotoxic chemotherapy, such as folinic acid/5-FU/oxali platin, folinic acid/5-FU/irinotecan, capecitabine/oxaliplatin, or folinic acid/5-FU/oxaliplatin/irinotecan. The addition of anti-VEGF therapy to these regimens has also been shown to improve survival and has the addition of anti-EGFR therapy in patients with wild-type RAS disease.2

The liver is the most common site of colorectal cancer metastasis, and the occurrence of liver metastases has a significant impact to patient prognosis. Therefore, effective treatment of colorectal liver metastases (CRLM) is key to improving patient survival rates and quality of life. Radical surgery is recognized as the best treatment for CRLM. However, the conversion rate for CRLM is not high, only 20–40%, and the postoperative recurrence rate is over 70%. Most recurrences of CRLM occur within 2 years post-surgery.3 Current clinical guidelines, both domestically and internationally, recommend that resectability of CRLM based on the premise of radical surgery R0 resection. However, scattered tumor cells, also known as minimal residual disease (MRD), can remain after primary tumor resection, and can be the main factor in tumor recurrence.3 Total neoadjuvant therapy (TNT), an emerging treatment modality that involves completion of all treatment modalities, including both chemoradiotherapy and chemotherapy prior to transabdominal resection, is now recognized as the preferred standard for the treatment of locally advanced rectal cancer. Its advantages include higher rates of pathologic complete response (pCR) rates and longer disease-free survival (DFS), minimizing the length of time patients need an ileostomy, facilitating resection, and improving the tolerance and completion rates of chemotherapy.4 Pelvic cavity magnetic resonance imaging (MRI) is the recommended diagnostic procedure for rectal cancer staging. MRI-diffusion weighted imaging (DWI) can help to distinguish post-treatment changes from residual active tumor using apparent diffusion coefficient (ADC) values. It is recognized as a non-invasive method for monitoring the response to preoperative radiochemotherapy.5

Limitations in the treatment of advanced rectal cancer primarily stem from the insufficient individualized treatment for different patients, resulting in poor efficacy and limited responses in some patients to existing targeted therapies and immunotherapies, which restrict treatment options. ACT has become the fourth treatment following surgery, chemotherapy, and radiotherapy. ACT typically includes cytokine-induced killer (CIK) cells,6,7 dendritic cells (DC)-CIK,8,9 cytotoxic T lymphocytes (CTL),10 tumor-infiltrating lymphocytes (TIL),11 and natural killer (NK) cells.12,13 These cell therapies are designed to use the body’s natural defense mechanisms to target and eliminate cancer cells and usually have fewer side effects or risks. On the other hand, cell therapies based on chimeric antigen receptor (CAR) technology have emerged, such as CAR-T cell, CAR-NK, or CAR macrophages (CAR-M), with typically utilize either autologous stem cells, allogeneic or xenogeneic cells, or genetically modified cells that require higher levels of manipulation and are considered high risk.

CIK cells possess a high proliferation rate and potent antitumor effects with MHC-unrestricted cytotoxicity. CIK cells are heterogeneous immune effector cells with a mixture of T cell and NK cell-like phenotypes, making them a type of T cell at the interface between the innate and adaptive immune systems.14 These are not restricted by MHC and have broad-spectrum anti-tumor and viral effects. The clinical trials of CIK cells (or CIK cells combined with DCs) have exceeded 80 globally for a variety of solid tumors, such as lung cancer, breast cancer, brain cancer, and colon cancer. Alongside other immunotherapies, conventional chemotherapies, or radiot herapies, CIK therapy suggested a synergistic anti-tumor effect.15 NK cells are the primary effector cells of the innate immune system. They can non-specifically kill tumor cells and virus-infected cells without prior sensitization.16 The abundance and function of NK cells are associated with clinical outcomes in various tumors, as NK cells not only participate in cancer immune surveillance but also support treatment responses induced by various therapies, including chemotherapy, radiotherapy (RT), targeted anticancer drugs, and peptide-mediated oncolysis. Oncological treatments, particularly chemotherapy, attenuate both the abundance and the function of patient’s endogenous NK cells. Those factors support the therapeutic principle of adoptive NK cell transfer. Currently, over 40 clinical trials are ongoing to evaluate the safety and efficacy of adoptively transferred NK cells (often combined with other therapeutic modalities) in patients with solid tumors, but signals of efficacy remain sporadic.17 Most of these studies are on allogeneic NK cell therapy, because unlike cytotoxic T cells, their MHC independence does not trigger Graft vs Host Disease (GvHD). However, autologous cells have become a promising strategy when allogeneic NK cells are limited or unavailable.

The inevitable resistance to chemotherapy, narrow mechanisms of action and toxicity, limit its efficacy. Multiple studies have shown that chemotherapy combined with immune cell therapy prolongs the overall survival (OS) and disease-free survival (DFS), improves immune function and quality of life, and reduces chemotherapy side effects.18,19 In this report, the combination of alternating autologous CIK cells and NK cells with standard first-line chemotherapy in TNT treatment mode has successfully treated advanced rectal cancer patients. The infusion of immune cells improves the immune function status of patients, achieving long-term control of tumors.

Case Presentation

The patient, a 57-year-old man was diagnosed as rectal cancer with synchronous liver metastasis stage IV (T4aN0M1) in September 2020 and underwent a radical resection of rectal cancer. The primary tumor measured 6.0 × 5.0cm. Histological examination revealed moderately differentiated adenocarcinoma with serosal invasion (Figure 1A and B). Vascular invasion was observed, and nerve infiltration was not. Immunostaining showed positive for MLHI, MSH2, MSH6, PMS-2, negative for cerbB2, and PD-L1 Tumor Proportion Score (TPS) <1%. Next-generation sequencing (NGS) results show HLA-C, TP53, APC, SMAD2, SOX9, ARID1A, PTCH1 gene mutations, KRAS, NRAS, BRAF, NTRK1, NTRK2, NTRK3 gene wild type, tumor mutation burden (TMB) 6.09, MSS. The images of contrast-enhanced CT showed masses in the 2nd segment of the left hepatic lobe and the 6th segment of the right hepatic lobe, with sizes of 2.3 × 2.0cm and 0.6 × 0.5cm, respectively (Figure 2A). The multidisciplinary team (MDT) meeting evaluated potential resectable liver metastases. Liver metastasis surgery can be performed after two cycles of translational therapy.

Figure 1 Histological and immunohistochemical staining results. Hematoxylin and eosin (HE) staining of the rectal lesions (A and B). HE staining of the 2nd segment of the left hepatic lobe lesions (C and D). HE staining of the 6th segment of the right hepatic lobe lesions (E and F). (A, C and E) magnification, ×40; (B, D and F) magnification, ×100.

Figure 2 Upper abdominal imaging examination. Liver metastases before combination therapy (A). After 2 cycles of treatment, the liver metastases significantly reduced in size (B). Imaging after 6 cycles of combined treatment and resection of liver metastases (C). Follow up imaging after 2 years of treatment (D). Red arrows indicate liver metastases.

The patient received 1 course of autologous CIK cells or 1 course of autologous NK cells intravenous infusion combined chemotherapy of XELOX [Oxaliplatin (130 mg/m2 intravenously on Day 1) and Capecitabine (1500 mg orally twice daily on Days 1–14)] every 3 weeks (Figure 3A). A total of 40 mL of peripheral blood was collected from patients 1–3 days prior to chemotherapy. PBMCs were isolated and enriched by Ficoll density gradient centrifugation. After 14 days of culture, the cells were harvested and resuspended in Ringer’s lactate solution (100 mL) supplemented with 0.1% human serum albumin (HSA). Their phenotype was analyzed by flow cytometry method (Figure 3B). Each cycle can obtain over 10 × 109 CIK cells or over 6 × 109 NK cells. Routine laboratory parameters (differential blood counts, RBC parameters, white blood cell counts), blood chemistry (creatinine, AST/SGOT, ALT/SGPT, γ-GT, and LDH), and serum tumor markers of the digestive system were determined after each treatment and during the follow-up period. The CT imaging showed that the liver lesions were reduced by 47%, achieved PR according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (Figure 2B). The patient underwent a hepatic metastasis resection on December 30, 2020. The histological examination showed that the larger nodule was hepatocellular nodular hyperplasia with atypical hyperplasia (Figure 1C and D), and the smaller one was metastatic adenocarcinoma originating from the gastrointestinal tract (Figure 1E and F). The results indicate that the two cycles of translational therapy were successful.

Figure 3 Schematic diagram on schedules of CIK/NK cell treatments (A) and immunophenotypes monitoring (B).

After surgery, the patient continued to receive 4 cycles of autologous CIK/NK cells combined with 6 cycles of XELOX [Oxaliplatin (100mg/m2 intravenously on Day 1) and Capecitabine (1500 mg orally twice daily on Days 1–14)]. He received a total of 6 cycles of treatment for 4.5 months starting on November 12, 2020 (Figure 3A). Follow up and contrast-enhanced CT examination showed no tumor metastasis or recurrence (Figure 2C and D).

A total of 6 cycles of ACT and 8 cycles of chemotherapy were completed. Tumor markers were within the normal limits for carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 199, CA242, CA50, and CA274 (Figure 4). During treatment, the patient experienced one episode of mild nausea and one episode of grade 2 granulocytopenia. According to the EORTC Core Quality of Life Questionnaire (QLQ-C30), the patient’s quality of life improved after treatment (Table 1). No complications related to CIK/NK cells infusion were observed.

Table 1 Evaluation of the Patient’s Quality of Life Before and After Treatment

Figure 4 Blood tumor markers examination. All markers were maintained at a low lever before and after treatments, showing no difference in the level of CEA (A), CA199 (B), CA242 (C), CA50 (D) and CA724 (E). In these graphs, checking point “Bas” means baseline, while checking points “C1” - “C6” indicate after the first time to the sixth ACT.

Plasma was obtained after centrifugation of peripheral blood (10 min at 3000rpm). Aliquots (300 µL) were prepared and directly stored at −80°C. Plasma cytokines and lymphocyte subpopulations were determined by flow cytometry on a FACSCanto II instrument (BD Biosciences, San Jose, CA, USA). The number of lymphocytes declined rapidly in the Cycle 1 and Cycle 2 treatment, including total lymphocytes (Figure 5A), CD3+ T cells (Figure 5B), CD4+ T cells (Figure 5C), CD8+ T cells Figure 5D), NK cells (Figure 5E) and NKT cells (Figure 5F). After 2 to 3 cycles of chemotherapy combined with ACT, there was a significant recovery. Throughout the treatment, the expression of regulatory T cells (Tregs) (Figure 5G) and immune checkpoint PD-1 molecule gradually (Figure 5H) decreased, while the proportion of cytotoxic CD8+CD28+ T cells (Figure 5I) significantly increased. IFN-γ and IL-2 increased during treatment and returned to baseline levels after treatment (Figure 5J), whereas TNF-α and IL-10 showed a downward trend (Figure 5K). IL-6 was much higher than the other cytokines and continued to increase (Figure 5K). Overall, Th2-type cytokines showed predominant expression. The patient received regular follow-up examinations. As of now, the patient remains in NED. These results suggest that autologous CIK/NK cells combined with chemotherapy were safe and effective for this patient with advanced rectal cancer.

Figure 5 Changes in immunophenotyping and cytokines during treatment. There are total lymphocytes (A), CD3+ T cells (B), CD4+ T cells (C), CD8+ T cells (D), NK cells (E), NKT cell (F), Tregs (G), CD3+PD-1+ cells (H), CD8+CD28+ (I), Th1 cytokine (J), and Th2 cytokine (K). In these graphs, checking point “Bas” means Baseline, while checking points “C1” - “C6” indicate after the first time to the sixth ACT.

Discussion

This is a successful case of cellular immunotherapy combined with chemotherapy for patients with MSS liver metastatic rectal cancer. Approximately 70% of patients with stage IV colorectal cancer had synchronous CRLM.20 Research statistics among 977 patients with localized liver diseases, underwent CRLM resection rate is 35%. In this case, the patient’s liver metastases decreased by 47% and was given the opportunity for surgery after 2 cycles of treatment. The pathological results of liver metastasis surgery showed that there were no tumor cells in the larger mass. The tumor regression grade (TRG) was 0 which meant the larger lesion of liver metastasis had reached pathological complete response (pCR). The evaluation results of CT image and pathology strongly suggested that the appropriate combination of autologous CIK/NK cells and chemotherapy could have significant effects.

The vast majority of patients with metastatic colorectal cancer are MSS, with microsatellite instability (MSI)-H patients accounting for only 5%. It is usually considered that MSI-H type cancer is immune-inflamed type, whereas MSS type is immune-exclude or immune desert type. MSS CRC patients have a poor prognosis and are generally ineffective with single immunotherapy.21,22 Those patients typically have fewer tumor-infiltrating lymphocytes than those of MSI-H CRC patients, such as cytotoxic cells, CD8+, Th1, Th2, follicular T helper cells, and T cells. Intratumoral T cells can be replenished with fresh, non-exhausted replacement cells from sites outside the tumor.23 The increase in peripheral lymphocytes may contribute to the infiltration of lymphocytes within the tumor. ACT may alleviate lymphopenia caused by chemotherapy. In this case report, the patient’s lymphocyte depletion caused by chemotherapy was significantly restored after 2 to 3 cycles of ACT. Regulatory T cells (Tregs) are usually recognized as a specialized subset of CD4+ T cells function in the establishment and maintenance of immune tolerance.24 They constitutively express T lymphocyte antigen 4 (CTLA-4) and compete with CD28 to bind to CD80/CD86 (CTLA-4 has a higher affinity for CD80/CD86 than CD28),25 thereby activating the secondary signal of T cells and reducing the anti-tumor activity of effector T cells. During the treatment, the patient showed a decrease in the proportion of CD4+CD25+Foxp3+ Tregs and an increase in the proportion of cytotoxic CD8+CD28+ cells. Meanwhile, the expression of immune checkpoint PD-1 molecules on the surface of T cells was reduced.

Th1 cytokines play a vital role in the anti-tumor immune response, while Th2 cytokines play an anti-Th1 response role in the process.26 The patient exhibited lower levels of Th1 cytokines than those of Th2 cytokines, and immune balance shifts to Th2. This situation is generally seen in cancer patients. Studies have found that the secretion of IL-2 and INF-γ in peripheral blood of the patients with advanced tumors decreased, and the secretion of IL-10 increased. Th2 cell drift is positively correlated with the malignancy of tumors, such as breast cancer,27 cervical cancer,28 melanoma,29 etc. The patient’s IL-2 and INF-γ levels increased, while IL-4 and IL-10 levels decreased slightly during treatment. However, the patient’s IL-6 level continues to increase. It suggests that the cell therapy has played a certain role in regulating Th1/Th2 balance, but this effect is limited. The above changes in the patient’s immune parameters suggest that CIK/NK cell immunotherapy may improve the patient’s immune function and alleviate immune suppression.

The immune function of the body is closely linked to the occurrence, development, and recurrence of tumors. ACT can not only exert anti-tumor effects but also improve or rebuild immune function to restore immune surveillance function, thereby preventing tumor recurrence. Research has shown that the recurrence rate within 12 months of the CRLM surgery was as high as 44.7%.30 The existing research is focused on the combination of CIK cells alone with or NK cells alone with radiotherapy and chemotherapy. In order to improve the responsiveness of the ACT, we adopted a treatment plan of alternating infusion of CIK cells and NK cells, and achieved good outcome. The patient has been in the NED state for 40 months and will continue to maintain this state. In addition, ACT could alleviate pain and improve quality of life.31 His quality of life has significantly improved.

In addition to effectiveness, this regimen has an excellent safety profile. During treatment, one episode of mild nausea and one episode of degree 2 granulocytopenia were reported, with no adverse reactions related to the ACT. Although CIK and NK are not able to recognize tumor-specific antigen-like CAR technology and TIL cells, they are safer, more practical and more cost-effectiveness. However, this treatment plan has limitations, including variable efficacy depending on individual patient differences, and the potential for immune-related side effects. Its main side effects are mainly low-grade fever, as well as chills, rashes, fatigue, headaches, etc. Therefore, in clinical applications, it is necessary to comprehensively consider the overall situation of the patient and develop personalized treatment plans, such as monitoring the patient’s immune status during the treatment process.

Conclusion

In summary, the success of the case provides a new first-line treatment for advanced MSS type rectal cancer, especially APC gene mutations and KRAS, NRAS, BRAF gene wild type. We have also achieved good clinical results with cell therapy alone and chemotherapy without the use of targeted drugs. The advantages of this treatment method include minimal side effects and a high level of general applicability, particularly in its ability to achieve long-term tumor control by improving the patient’s immune status. However, further research and clinical trials are needed to verify its efficacy and populations that benefit from it. At present, early activation of the immune system may be a factor in screening the types of beneficiaries.

Ethics Approval and Informed Consent

Studies involving human participants were reviewed and approved by the Ethics Committee of the Northern Theater Command General Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. As reported in the case, the patient showed improvement in multiple immune indexes after 2 to 3 cycles of treatment.

Consent for Publication

The authors have obtained informed consent from the patients for publication of the case details and any accompanying images, and the ethics committee of The General Hospital of Northern Theater Command approved this consent process and the publication of case details.

Funding

This study was supported by a grant from the Natural Science Foundation of Liaoning Province (Grant Number: 2020JH2/10300160).

Disclosure

The author(s) report no conflicts of interest in this work.

References

1. Rebecca LS, Nikita SW, Andrea C, et al. Colorectal cancer statistics, 2023. CA Cancer J Clin. 2023;73(3):233–254. doi:10.3322/caac.21772

2. Daenielle L, Kristen KC. Diagnosis and management of rectal cancer in patients younger than 50 years: rising global incidence and unique challenges. J Natl Compr Canc Netw. 2022;20(10):1169–1175. doi:10.6004/jnccn.2022.7056

3. Traci LH, Victor MZ. Management of synchronous colorectal cancer metastases. Surg Oncol Clin N Am. 2022;31(2):265–278. doi:10.1016/j.soc.2021.11.007

4. Al BB, Alan PV, Mohamed A, et al. NCCN guidelines® insights: rectal cancer, version 3.2024. J Natl Compr Canc Netw. 2024;22(6):366–375. doi:10.6004/jnccn.2024.0041

5. Daniela M, Francesca DF, Anna LM, et al. Diffusion-weighted magnetic resonance application in response prediction before, during, and after neoadjuvant radiochemotherapy in primary rectal cancer carcinoma. Biomed Res Int. 2013;2013:740195. doi:10.1155/2013/740195

6. Wanjun Y, Fei Y, Xiao Y, et al. A Phase I/II clinical trial on the efficacy and safety of NKT cells combined with gefitinib for advanced EGFR-mutated non-small-cell lung cancer. BMC Cancer. 2021;21(1):877. doi:10.1186/s12885-021-08590-1

7. Erika F, Alessandra M, Lorenzo D, et al. Integrated antitumor activities of cellular immunotherapy with CIK lymphocytes and interferons against KIT/PDGFRA wild type GIST. Int J Mol Sci. 2022;23(18):10368. doi:10.3390/ijms231810368

8. Shuo W, Xiaoli W, Xinna Z, et al. DC-CIK as a widely applicable cancer immunotherapy. Expert Opin Biol Ther. 2020;20(6):601–607. doi:10.1080/14712598.2020.1728250

9. Wei-Qiang T, Li Y, Xu C, et al. Overexpression of lncRNA TUG1 enhances the efficacy of DC-CIK immunotherapy in neuroblastoma in vitro and in vivo. Cancer Biomark. 2023;36(1):53–61. doi:10.3233/CBM-210436

10. Bagher F, Masoud N, Keywan M. CD8 + cytotoxic T lymphocytes in cancer immunotherapy: a review. J Cell Physiol. 2019;234(6):8509–8521. doi:10.1002/jcp.27782

11. Benjamin CC, Chao W, Jamie KT, et al. Tumor-infiltrating lymphocyte treatment for anti-PD-1-resistant metastatic lung cancer: a Phase 1 trial. Nat Med. 2021;27(8):1410–1418. doi:10.1038/s41591-021-01462-y

12. Melissa AG, Sarah C, Patricia LJ, et al. A Phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer. Cytotherapy. 2011;13(1):98–107. doi:10.3109/14653249.2010.515582

13. Jacob AM, Jeffrey SM. Exploring the NK cell platform for cancer immunotherapy. Nat Rev Clin Oncol. 2021;18(2):85–100. doi:10.1038/s41571-020-0426-7

14. Vincenzo C, Mitch K. The role of invariant NKT cells at the interface of innate and adaptive immunity. Semin Immunol. 2010;22(2):59–60. doi:10.1016/j.smim.2010.01.002

15. Jia H, Bowen Z, Senyu Z, et al. The progress and prospects of immune cell therapy for the treatment of cancer. Cell Transplant. 2024;33:9636897241231892. doi:10.1177/09636897241231892

16. Sizhe L, Vasiliy G, Yekaterina G, et al. NK cell-based cancer immunotherapy: from basic biology to clinical development. J Hematol Oncol. 2021;14(1):7. doi:10.1186/s13045-020-01014-w

17. Le T, Carlos J, Apple T, et al. NK cells and solid tumors: therapeutic potential and persisting obstacles. Mol Cancer. 2022;21(1):206. doi:10.1186/s12943-022-01672-z

18. Wang AX, Ong XJ, D’Souza C, et al. Combining chemotherapy with CAR-T cell therapy in treating solid tumors. Front Immunol. 2023;14:1140541. doi:10.3389/fimmu.2023.1140541

19. Pocaterra A, Catucci M, Mondino A. Adoptive T cell therapy of solid tumors: time to team up with immunogenic chemo/radiotherapy. Curr Opin Immunol. 2022;74:53–59. doi:10.1016/j.coi.2021.10.004

20. Mustafa R, Sidra H, Philip HGI, et al. Liver resection improves survival in colorectal cancer patients: causal-effects from population-level instrumental variable analysis. Ann Surg. 2019;270(4):692–700. doi:10.1097/SLA.0000000000003485

21. Amir TE, Seyed YS, Ehsan NM, et al. MSI-L/EMAST is a predictive biomarker for metastasis in colorectal cancer patients. J Cell Physio. 2019;234(8):13128–13136. doi:10.1002/jcp.27983

22. Sanghee K, Younghyun N, Sung YJ, et al. The significance of microsatellite instability in colorectal cancer after controlling for clinicopathological factors. Medicine. 2018;97(9):e0019. doi:10.1097/MD.0000000000010019

23. Thomas DW, Shravan M, Patricia EA, et al. Peripheral T cell expansion predicts tumour infiltration and clinical response. Nature. 2020;579(7798):274–278. doi:10.1038/s41586-020-2056-8

24. Iris KG, Daniel JC. Organ-specific and memory treg cells: specificity, development, function, and maintenance. Front Immunol. 2014;15:333. doi:10.3389/fimmu.2014.00333

25. Navid S, Dana RT, Aram D, et al. CTLA-4 in regulatory T cells for cancer immunotherapy. Cancers. 2021;13(6):1440. doi:10.3390/cancers13061440

26. Peña-Romero AC, Orenes-Piñero E. Dual effect of immune cells within tumour microenvironment: pro- and anti- tumour effects and their triggers. Cancers. 2022;14(7):1681. doi:10.3390/cancers14071681

27. Yu X, Yi H, Jianping J, et al. Identification of the prognostic value of Th1/Th2 ratio and a novel prognostic signature in basal-like breast cancer. Hereditas. 2023;160(1):2. doi:10.1186/s41065-023-00265-0

28. Teng F, Cui G, Qian L, et al. Changes of T lymphocyte subsets in peripheral blood of patients with intermediate and advanced cervical cancer before and after nimotuzumab combined with chemoradiotherapy. Int Arch Allergy Immunol. 2023;184(1):85–97. doi:10.1159/000525487

29. Gérard A, Doyen J, Cremoni M, et al. Baseline and early functional immune response is associated with subsequent clinical outcomes of PD-1 inhibition therapy in metastatic melanoma patients. J Immunother Cancer. 2021;9(e002512):e002512. doi:10.1136/jitc-2021-002512

30. Sternschuss M, Goshen-Lago T, Perl G, et al. Characterization of biomarkers in colorectal cancer liver metastases as a prognostic tool. J Pers Med. 2021;11(11):1059. doi:10.3390/jpm11111059

31. Moon JC, Jeong YP, Seungmin B, et al. Phase II clinical trial of ex vivo-expanded cytokine-induced killer cells therapy in advanced pancreatic cancer. Cancer Immunol Immunother. 2014;63(9):939–946. doi:10.1007/s00262-014-1566-3