Colorectal cancer (CRC) is the third most common cancer and the second cause of cancer death globally (1). CRC is a complex, molecularly heterogeneous disease that is characterized by diverse genomic and immunologic landscapes. It shows different incidence, pathogenesis, molecular pathways, immunogenicity, and patient outcome depending on tumor location (2, 3). The inherent complexity of these multifactorial diseases dramatically complicates predicting patients’ prognoses and responses to treatment (4). AJCC/UICC-TNM is a standard method for the classification of malignant tumors. This method relies on tumor characteristics, including primary tumor extension (T), lymph node involvement (N), and distant metastasis (M)(6). Despite global acceptance, the importance and power of TNM-staging, it has some drawbacks as patients in the same stage, could have different clinical outcomes and prognosis (5, 6).

Nowadays, it is extensively accepted that the immune components of the tumor microenvironment (TME) play a critical role in tumor development (7). Accordingly, analysis of the interactive relationships between tumor cells and the immune system components in the TME have received more attention (8). Many studies have established that the high density of different T cell subpopulations such as CD3+ T cells, CD8+ T cells, and CD45RO+ memory T cells in tumor tissue is associated with more prolonged overall survival (OS) and disease-free survival (DFS) in different tumor types (8).

One of the critical regulatory molecules in the TME is immune checkpoints (ICPs) that regulate the functions of infiltrated immune cell. ICPs refer to both activatory and inhibitory molecules which act as gatekeepers of immune responses. The inhibitory checkpoints include, but are not limited to programmed cell death receptor 1 (PD-1), cytotoxic T-lymphocyte associated protein-4 (CTLA4), T cell immunoglobulin domain and mucin domain-containing-3 (Tim3), Lymphocyte Activating 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and B- and T-lymphocyte attenuator (BTLA) and. Their expression in malignant tumors is often significantly increased and is associated with poor prognosis (9). But, little is understood about how comprehensive regulation patterns of ICP molecules relate to immune responses, TME formation, and patient outcomes (10).

PD-1 belongs to the immunoglobulin superfamily and is expressed on the activated and regulatory T cells (Treg), B cells, natural killer (NK) cells, NKT cells, macrophages, dendritic cells, and monocytes (11, 12). The engagement of PD-1 by its ligands, PD-L1 and PD-L2, induces tumor immune escape and down-regulation of tumor-infiltrating lymphocytes (TILs) by different mechanisms such as: inhibiting T cell proliferation and induction of their apoptosis, reducing the inflammatory cytokines as IFN-γ, IL-2, TNF-α, inhibiting granular enzyme and perforin production by cytotoxic T lymphocytes (CTLs), and increasing metastasis and penetration of tumor cells (12–14). Immune checkpoint inhibitors (ICIs) targeting PD-1 or PD-L1 have shown objective responses in certain cancers, including melanoma, renal cell carcinoma, and non-small cell lung cancer (15). Pembrolizumab and nivolumab, as PD-1-blocking antibodies, have also been accelerated FDA approval after showing their effectiveness in the patients with metastatic CRC (16). While PD-1 showed inhibitory and therapeutic effects, there are pieces of conflicting evidence on its prognostic significance in CRC (17–23).

Tim3is another immunoglobulin superfamily co-inhibitory receptor expressed on both immune and tumor cells, including CTLs, type 1 T helper (Th1) cells, Th17, Tregs, and innate immune cells (24). Described ligands for Tim3 are high-mobility group protein B1 (HMGB1), galectin 9, carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), and phosphatidylserine (PtdSer) (25). Even though the Tim3 intracellular signaling pathways have not been fully elucidated, it appears that they finally lead to inhibiting antitumor immune responses by blocking effector T cell responses, increasing Treg functions, and growing myeloid-derived suppressor cells (MDSCs) inside tumors (26–28). A growing number of studies have shown that Tim3 up-regulation in tumor tissue is associated with poor prognosis in a wide variety of cancers such as CRC, gastric cancer, prostate cancer, clear cell renal cancer, urothelial bladder cancer, and cervical cancer (29–34). Thus, Tim3 could be introduced as a potential prognostic factor and a new target for immunotherapy in solid tumors (35). In CRC, Tim3 expression has been reported to be higher in tumor tissues than in normal tissues and is significantly associated with advanced stages and metastasis (36). However, the prognostic value of Tim3 expression in tumor tissue or immune cells in patients with CRC isn’t well documented (20, 29, 37). Previous studies have shown that though Tim3 expression is associated with T cell exhaustion in cancer patients, the co-expression of Tim3 with PD-1 represents more exhausted CD4+ and CD8+ T cells (38, 39). Consistently, preclinical studies demonstrated that co-blockade of Tim3 and PD-1 pathways is highly effective in the treatment of solid tumors (40).

Accordingly, we hypothesized that the PD-1 and Tim3 expression could be associated with poor prognosis and their co-expression would be a robust prognostic marker that besides the TNM staging system can be useful in dividing patients into more homogeneous groups. Moreover, according to the different characteristics of the right and left-sided CRCs, the prognostic impact of PD-1 and Tim3 would be different on each tumor side. Therefore, in this study, we also examined the expression of these markers in the center (CT) and invasive margin (IM) of tumor tissues in a total cohort and subdivided left and right-sided CRCs to determine whether they have any relations with clinicopathological parameters and prognosis.

The fundamental, undeniable role of the immune system in tumor evolution and progression has prompted the evaluation of immune cell infiltration, as a prognostic marker for solid tumors, including CRC (41). Studies have shown that high infiltrations of CD3+ and CD8+ T cells in the TME are associated with improved DFS and OS in patients with CRC (8). Accordingly, in the present study, we assessed the CD3 and CD8 expressions in the IM and CT to evaluate their prognostic potential in CRC, however similar to Barbosa et al. (42), no statistically significant association was found in the total cohort. Since proximal and distal CRC displays distinct epidemiological, pathological, and clinical entities (2, 3), we further analyzed TILs infiltration and their association with clinical outcomes based on the tumor location. Repeating the analysis in the right and left-sided CRCs, showed that the CD8 expression in IM of right-sided CRCs was significantly associated with improved OS. No significant association between T cell infiltration and survival might imply that T cells fail to eliminate cancer cells in TME effectively. There are a wide variety of immunosuppressive mechanisms in the TME that can result in modulation and functional impairment of T cells. In the TME, infiltrated immune cells display a broad spectrum of dysfunctional states that are shaped by suppressive signals within the TME (43, 44). The role of these immune cells in tumor growth and progression is diverse and is tightly linked to the molecules and ligands they express (41). Among them, up-regulation of inhibitory ICPs is a hallmark of the tumor ecosystem resulting in immune cell dysfunction (9).

PD-1 is primarily introduced as an inhibitory receptor for the immune system, and its inhibition has been reported to restore immune activities in several malignancies (15). Nevertheless, some studies showed that PD-1 expression had been linked to favorable prognosis in several malignancies, including gastric, breast, small cell lung cancer, HPV-associated head and neck cancer, and CRC, as well (37, 45–49). Accordingly, the effect of this receptor on T cell function and tumor prognosis remains unclear. The results of our study showed that higher PD-1 expression in the IM was associated with lower T-stage (T1/2) in the total cohort and left-sided CRCs, lower M-stage (M0) in the total cohort and right-sided CRCs, lower TNM stage (I/II) just in the total cohort, lack of metastasis in the total cohort and both right and left-sided CRCs, lack of recurrence just in left-sided CRCs, and conversely with larger tumor size (≥5 cm) just in right-sided CRCs. Consistently, Saleh et al. showed that PD-1 gene expression in both tumor tissue and blood circulation of patients with CRC is higher in the early stages of CRC (50). Higher expression of PD-1 in the early stages of the CRC, could implies that targeting PD-1 in these stages might be more effective.

The prognostic association of PD-1 expression in CRC has been also examined in several studies, however the results are inconsistent (17, 18, 23). Controversial results could be depending on some parameters such as tumor stage, location, or mismatch repair (MMR) status. Zengin et al. showed that high PD-1 expression could be an independent poor prognostic factor for recurrence-free survival (RFS) and OS in CRC patients in stage III (23). Contradictory, Li et al. showed that high PD-1 expression in TILs is an independent prognostic factor for improved OS and DFS in CRC patients, particularly for MMR-proficient subgroup (49). Lee et al. showed that the high PD-1 expression in TILs in MMR-deficient tumors is relined to enhanced RFS only when PD-L1 expression in tumor cells is low (21). Furthermore, Ahtiainen et al. found that having a high density of PD-1+ TILs was linked to better OS and DFS in CRC, regardless of the MMR status or immune cell score (17). Berntsson et al. showed that high PD-1 expression on immune cells is associated with improved OS, and this association depends on tumor location (18). These observations call into question the prognostic value of PD-1 in CRC. Similarly, our results showed that high PD-1 expression in the IM was associated with better OS; however, it was not statistically significant. Nevertheless, we observed that classifying patients based on their CD8 expression and tumor location, high PD-1 expression in IM was associated with improved OS only in the CD8 high expression subgroup.

Due to similar effects on survival, we next incorporated CD8 and PD-1 expressions patterns into a single score to investigate whether they might work together to improve prognostic power. As a result, prognostic models demonstrated that patients with high expressions of PD-1 and CD8 in the IM had better OS than other patients; however, it was not significant. This result indicated that PD-1+ CD8+ TILs had proper effector function in the IM of CRC.

The association between high PD-1 expression (with or without CD8) and better OS and lower stage of the disease is in contrast with the commonly defined role for this molecule as PD-1 generally is introduced as an inhibitory receptor and is a hallmark of T cell exhaustion. Due to the low PD-L1 expression in CRC tumors, it can be concluded that in the absence of PD-1 ligation to its ligand, the high expression of PD-1 in the TILs might reflect the effector phenotypes of these cells (21, 51). It has recently been demonstrated that PD-1 differentially affects cell proliferation, maturation, and transcript signatures among diverse immune cell populations. While naive T cells are inhibited following PD-1 ligation, T cells with effector and central memory phenotypes proliferate after ligation of PD-1 by its ligand (52). There is also evidence that PD-1 signaling is not essential for CD8+ T cell exhaustion (53). Moreover, recent studies have demonstrated that CD8+ T cells remain functional despite PD-1 expression in different tumors such as breast cancer, non-small cell lung cancer, and gastric cancer (54–56).

Tim3 is another inhibitory receptor that has been reported as a negative prognostic factor for tumors such as CRC, alone or in combination with PD-1 (20, 35). Little is known about the prognostic role of Tim3 in cancer. While Tim3 was associated with worse prognosis in non-small cell lung cancer and gastric cancer, it corelated with a better prognosis in breast cancer (57). To the best of our knowledge, this is the first study that evaluated the Tim3 expression on the immune cells in the IM and CT, according to the primary site of the tumor in CRC. our results indicated that, Tim3 expression was just observed on immune cells (innate and adaptive), while other studies on CRC reported its expression on tumor cells, as well (20, 29). We also observed that Tim3 expression is upregulated in tumor tissue comparing to normal-like adjacent tissue, and this expression in the CT was associated with higher M-stage (M1) in left-sided CRCs, older age in the total cohort, and left-sided CRCs. In addition, high expression of Tim3 in CT was associated with poor OS in the total cohort and left-sided CRCs (especially in patients with high infiltration of CD8+ TILs). In the adjusted model, Tim3 in CT remained an independent poor prognostic factor besides T-stage, M-stage, lymph node involvement, and tumor budding. These results are in concordance with previous studies showing that Tim3 expression is upregulated in tumor tissues and is associated with poor prognosis in patients with CRC, gastric cancer, prostate cancer, clear cell renal cancer, urothelial bladder cancer, and cervical cancer (29–34).

Regarding Tim3 expression on immune cells, previous studies on gastric cancer showed that Tim3 expressions on T cells (CD8+ and Treg) and NK cells, respectively, were associated with poor prognosis and advanced stages of the tumor (58, 59). Moreover, increased Tim3 expression on tumor-specific CD8+ T cells was associated with impaired CD8+ T cell function and poor prognosis in HBV-associated hepatocellular carcinoma (60) and prostate cancer (61). In CRC, it is reported that Tim3+CD8+ T cells are more prone to apoptosis than Tim3- CD8+ T cells (62). In addition, upregulation of Tim3 and PD-1 on CD8+ and CD4+ T cells is associated with the dysfunctionality of these cells and less IFNγ production (39). All of these studies indicate that overexpression of Tim3 in TME has inhibitory effects on immune responses against tumors. Paradoxically, Al-Badran et al. showed that Tim3 expression on stromal immune cells is associated with a better CRC prognosis (37). Similarly, we observed that the Tim3 expression on immune cells at the IM was higher in patients who had no metastasis and were alive.

To investigate the effects of PD-1 and Tim3 expression in different regions (CT and IM) on survival, we combined their expression levels in the IM and CT. This combination for Tim3 showed that patients with high expression of Tim3 in the CT while low expression in the IM, had significantly a poorer OS than other groups. These data indicated that the distribution of Tim3+ immune cells in the tumor could probably reflect different functions or differentially affect clinical outcomes and prognosis. The difference might be explained by the difference in the expression of Tim3 ligands such as galactin-9 in different tumor areas. On the other hand, this combination for PD-1 showed that patients with high expression of PD-1 in IM but low expression in CT had a better OS than other groups; however, it was not significant. This issue should be a topic that warrants further investigation in a larger population.

To evaluate the synergistic effect of these two inhibitory ICPs on survival, we combined their expression in CT and IM and repeated the analysis. We observed that patients with high expression of both PD-1 and Tim3 in IM but low PD-1 and Tim3 expression in CT had improved OS; however, their relations weren’t statistically significant, may be due to the small sample size. In this regard, Al-Badran et al. similarly showed that the combination of high expression of Tim3, LAG-3, and PD-1 on the stromal immune cells is associated with better outcomes in CRC patients (37). Contradictory, Kuai et al. showed that co-expression of PD-1 and Tim3 is associated with a worse prognosis in CRC (20). Based on these results and new data (63–65), PD-1 and Tim3 probably have a dual function, and some key factors affect the activity of these molecules in the TME. Thus, understanding the role of these molecules can be improved by identifying these key factors in the TME.

In conclusion, our findings suggest that the clinicopathological and prognostic significance of immune system-related markers such as CD8, PD-1, and Tim3 depends on the primary tumor sides. Such studies could potentially be helpful in patient clinical management, since our results suggest that the tumor side may be a factor in therapeutic decisions, including immunotherapy based on inhibitory receptors. We also noted that PD-1 and Tim3 expressions in different regions of the resected tumor have different prognostic impacts, which could be explained by the different properties of TME in each region. This finding should be more evaluated in future studies to confirm and determine the reasons for these differences. Overall, we showed that Tim3 could act as a prognostic factor and therapeutic target in CRC. This marker is probably a more preferred target for immunotherapy than PD-1, especially in left-sided CRCs.

The data used and/or analyzed during the current study are available in the CRC-ICM data set (66) (https://data.mendeley.com/datasets/h3fhg9zr47).

The studies involving human participants were reviewed and approved by school of medicine, Isfahan university of medical sciences (IR.MUI.MED.REC.1398.611). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

ZM and ST carried out the experiments. ZM wrote the main manuscript text and prepared figures. MS and AD carried out pathology sections analysis and data collection. ZM done data collection and statistical analysis. ZH helped in statistical analysis. ZF helped shape the research, analysis and manuscript edition. MR conceived the original idea and supervised the project and manuscript final edition. All authors contributed to the article and approved the submitted version.