In this study, from 24 patients with at least 2 years of follow-up there was only one patient with disease recurrence. This patient (male, 88 years old, 54 months of follow-up) has a rectal cancer stage II with a KRAS mutation and he was treated with radiotherapy and chemotherapy in addition to surgery. He displayed the lowest percentage of CD4 naïve T cells (6.1%), IgM memory B+ cells (30.7%), and switched memory B cells (1.1%) along with the highest percentage of DN B cells (32.5%) as compared to the other patients. The decrease in effector B cells along with the accumulation of inflammatory and senescent B cells (DN) can explain, at least in part, the disease recurrence in this patient. Effector B cells not only produce anti-tumor specific antibodies but also present tumor antigens to T cells leading to activation, clonal expansion, and memory T cells. In addition, B cells recognizing antigens in tumor cells produce granzyme and kill directly tumor cells. (28, 29, 30) In human CRC microenvironment, B cells infiltration has been associated with stage I and improved disease-specific survival. In addition, the high infiltration of B lymphocytes along with CD8 lymphocytes was associated with improved prognosis. (31) In another study, a high score infiltration of B cells/plasma cells showed a tendency towards prolonged survival whereas a high score of T cell infiltration was significantly associated with longer cancer-specific survival and lower disease stage. (32). However, there are few studies evaluating the peripheral circulating immune cells in CRC patients. Zhang et al. (16) showed that CRC patients with advanced stage of disease displayed decreased percentage of functional CD8 naïve and central memory (CM) T cells whereas terminally differentiated CD4 and CD8 T cells increased suggesting immunosenescence. In addition, no differences were found according to CRC stage on the B cell subsets. We found no significant differences in T and B subsets when CRC patients in stage I/II were compared with CRC patients in stage III. These results could be explained by the great clinical heterogeneity of this population since 6 patients in stage I/II were treated similarly to patients in stage III (chemotherapy or chemotherapy plus radiotherapy). In addition, it has to be considered that the tumor itself has immunosuppressive actions and chemotherapy/radiotherapy can also interfere with immune cell numbers and functions. Another important subset in cancer progression are MDSCs as they inhibit the anti-cancer effector activity of T and B cells and contribute for the development of T regulatory cells which can also suppress the immunity against tumor. (33, 34) MDSCs lead to an immunosuppressive tumor microenvironment and contribute for cancer progression. Surgery is the best therapy for removing the immunosuppressive microenvironment generated by the tumor mass, but some MDSCs can still be found in circulation after surgery and impair patient prognosis. It has been shown that gemcitabine or 5-fluorouracil (5-FU) can inhibit MDSCs but they can suppress bone marrow in addition to other side effects. (36, 37). In our study, it was not found significant difference in the median MDSC percentage between CRC patients stage I/II and stage III. However, one CRC stage III patient presented the highest percentage of MDSCs (9%) despite of the use of chemotherapy (5-FU), and one patient in stage I and using chemotherapy (5-FU) for nodules on the liver, presented the highest percentage of MDSCs (5.8%) in this group of patients.
Because angiotensin converting enzyme (ACE) has been shown to play a role in immunity, reproduction, and hematopoiesis in addition to the effects on blood pressure (22, 23, 38) we evaluated its expression in immune cells from CRC patients. Data from Surveillance, Epidemiology, and End-Results (SEER) showed that in 13,982 patients with CRC (65–84 years old, stages I to III), the use of ACE inhibitor (ACEi) was associated with decreased cancer-specific mortality. (39) In mice, the transfer of a colorectal cancer cell line caused liver metastases, and the administration of ACE inhibitor (captopril) reduced tumor viability and metastatic growth along with tissue increased infiltration of CD3+ T cells suggesting that the immune system can somehow be modulated by ACE (40). Moreover, patients in use of ACEi for hypertension and diagnosed with rectal cancer (stage II/III) presented a significant reduction in pathological staging after neoadjuvant radiation in comparison with the cohort of patients not taking these medications. (41) In metastatic colorectal cancer, patients using ACEi for hypertension and treated with bevacizumab showed a higher disease-free survival suggesting that ACEi could enhance the antiangiogenic effect of VEGF-a blockade. (42) In early-stage and left-sided CRCs, ACEi was associated with reduced risk of disease recurrence. (43) ACE expression in immune cells has been reported (19, 20, 21) and the overexpression of ACE in mice macrophages or neutrophils has been linked to a higher resistance to conditions that are common in old adults such as tumors, infections, chronic neurotoxicity and neuroinflammation. (23–27) However, the mechanisms associated to ACE actions in immune cells are still not completely understood. In our study, CRC patients in stage III exhibited a higher expression of ACE in CD4+ naïve and EM subsets whereas in CD8+ there was a trend towards higher expression of ACE in naïve, CM, and EM subsets in these patients. In B cells there was a trend towards higher expression of ACE in IgM memory and switched memory subsets in CRC patients stage III. We don’t know whether this increased expression of ACE in T and B cells from patients with more advanced tumor stage (III) has any beneficial or detrimental impact for the anti-tumor immune response. Interestingly, the studied patients using ACE inhibitor (two in stage II and one in stage III) as anti-hypertensive therapy presented the longest follow-up (156 months, 132 months, and 156 months, respectively). In addition, these patients presented a lower percentage of B switched memory cells expressing ACE than the median for each group (stage II median = 1.35%, patients = 0.9% and 0.5%; stage III median = 3%, patient = 1.2%). MDSCs expressing ACE did not show significant difference between CRC patients stage II/III and stage III and patients with the highest percentage of MDSCs were not using ACEi as anti-hypertensive therapy. These findings contribute for our hypothesis that ACEi interferes somehow with CRC patients’ outcome. (44). In addition, mice experiments have shown that ACE is associated with myeloid cells maturation and in turn interferes with MDSC generation. (45, 46)
Our group is the first to show the expression of ACE in T and B cells subsets as well as on MDSCs from CRC patients. We did not evaluate ACE genotypes, but it was found that the polymorphism of ACE expressed in tumor tissues is correlated with tumor size and patient survival in CRC. (47). On the other hand, two different groups found that in blood leukocytes ACE genotype is either associated with a risk of CRC (48) or not associated with increased risk of CRC (49).
In conclusion our results show that the expression of ACE is increased in Naïve and Effector T cells and in Effector B cells of CRC patients stage III. These findings open opportunities for targeting ACE in immune cells as a new therapy in colorectal cancer. Functional studies are required to answer whether ACE expression in T and B lymphocytes are correlated with patient prognosis in CRC.