Rectal cancer treatment depends on various factors, including clinical stage, pathological grade, and patient age. For patients who are diagnosed with locally advanced rectal cancer (LARC), preoperative 5-fluorouracil-based chemoradiotherapy (CRT), followed by surgery, has been recommended as the standard treatment (1). Neoadjuvant CRT (nCRT) has been demonstrated to reduce tumor size, make it easier to remove, and reduce the risks of local recurrence and distant metastasis (2). Importantly, pathological complete response (pCR), defined as no remaining visible tumor cells in the surgical specimen on a histopathologic assessment, has already been proven to be associated with prolonged disease-free survival (DFS) and overall survival (OS) (3). Unfortunately, the pCR ratio is only approximately 10-20% in LARC patients who receive nCRT, and most of the patients experience only mild to moderate tumor regression or even progression following nCRT (4). Numerous studies have been carried out to explore the mechanisms of the clinical response to chemoradiotherapy (5–7), but the biomarkers that represent nCRT sensitivity remain poorly understood; thus, further studies are needed to improve the pCR ratio of nCRT.

Recently, emerging evidence has shown that the antitumour effect of chemotherapy and radiotherapy is related to serious immune-related mechanisms (8). CRT was proven to improve the suppressive tumor microenvironment (TME) by suppressing or eliminating immunosuppressive cells (8, 9). as well as prompt tumor cells to release neoantigens, which results in the activation and proliferation of cytotoxic T lymphocytes (CTLs) (10). Respectively, the association between tumor-infiltrating immune cells (TIICs) and sensitivity of nCRT got extensive attention (11, 12). In breast cancer, circulating MDSC and Treg subsets were higher in non-responders than responders in patients after nCRT (9). Although studies above have attempted to investigate the role that TIICs play in the process of nCRT, their predictive value in LARC still needs to be confirmed through more studies.

The tumor immune microenvironment (TIME) is a spatially organized but complex ecosystem that is comprised of multiple immune cell types. To interrogate the complexity of the TIME, technologies were created to gauge the expression of multiple proteins within a single cell. However, for routine laboratory assays, such as flow cytometry, the tissue must be dissociated into a cell suspension, thus leading to the loss of architecture, which contains important bioinformatics information. This dilemma drove the development of imaging mass cytometry (IMC), which can solve the problems mentioned above (13, 14). IMC makes use of heavy metal-conjugated antibodies that are ionized from the surface of a tissue slide. Since rare-earth metals are not found in biological tissues, IMC circumvents the spectral overlap limitation in flow cytometry and allows a simultaneous distinguishment of over 50 parameters at the single-cell level. In addition, IMC retains spatial information, making it possible to reveal tissue context and cellular interactions that show distinct architecture in physiological and disease states. Because of its superior advantages, IMC has been popularly applied for the immunophenotyping of TMEs. This has greatly promoted the discovery of rare cell subsets and the assessment of the relationship between cellular phenotype diversity and therapeutic outcomes. Currently, IMC has been extended to mechanistic studies beyond phenotypic observations (15).

In the present study, to comprehensively assess the immune status of LARC and its correlation to clinical outcomes after nCRT, we developed a 12-antibody panel containing the major TIIC subsets (including CD4+ T cells, CD8+ CTLs, Tregs, NK cells, B cells, monocytes, and tumor-associated macrophages (TAMs)) for the analysis of LARC biopsy samples by IMC. Immunohistochemistry (IHC) and logistic regression analysis were adopted for independent validation in a larger group. The current study aimed to determine whether the density of infiltrating immune cells pre-nCRT was correlated with the subsequent treatment response and to allow more rational therapeutic strategies to be developed in the future.

In this study, we developed a 12-antibody panel containing the markers of the major TIIC subset to determine whether the immune status differs between LARC patients who respond to nCRT differently. A higher expression intensity of CD8 cells was observed in patients who achieved pCR, and a higher density of TAMs and Tregs was observed in those who did not. To further investigate the relationship between the densities of CD8+ CTLs, CD163+ TAMs and Foxp3+ Tegs in the local TME before nCRT and treatment response in LARC patients, a larger group of LARC patients were enrolled as an independent validation group. In the multiple logistic regression analysis, CTLs and TAMs were revealed as independent predictors for a good response to nCRT, suggesting that CTLs and TAMs play key immunoregulatory roles in RCT-associated antitumour processes. Therefore, CTL and TAM infiltration levels are expected to become potential predictive biomarkers of nCRT sensitivity in LARC patients.

Since recommended as the standard treatment for LARC, nCRT was proven to significantly improve pCR rates (1, 20). pCR after nCRT was proposed as a key prognostic indicator for better outcomes in LARC (3, 21). In a meta-analysis which included 1913 patients by Zorcolos et al, Patients with pCR had a much lower rate of local tumor recurrence and distant metastasis. Besides, OS was longer for those achieved pCR (92.9% for pCR versus 73.4% for non-pCR at 5^th year) (3). The similar results were seen in a German Rectal Trial (CAO/ARO/AIO-94 trial), 86% of patients with a pCR remained free of disease at the end of 5 years, whereas only 63% of patients without pCR (22). However, as in present study, only minority of LARC patients achieved pCR after received nCRT, up to 80% of the patients experienced incomplete tumor regression or even progression after nCRT (4). The current study was dedicated to explore the relationship of preoperative level of TIICs and efficiency of nCRT, and aid the therapeutic strategy-making.

In this study, we demonstrated that a higher CD8 + TIL count before nCRT was associated with a better pathologic response, which is reasonably explained by experimental results showing that CTLs play a crucial role in the antitumour effects of cytotoxic drugs and radiation (23, 24). Recently, it has been reported that several molecules, including calreticulin, HMGB1, and ATP, released by tumor cells are responsible for chemotherapy-induced anticancer immune responses (10, 25). These molecules mediate the proliferation and activation of CTLs via multiple pathways (26). Radiation therapy has also shown its antitumour effect by inducing neoantigens from tumor cells (10), similar to the role of chemotherapy, and this effect may be significantly weakened after the depletion of CTLs.

M1 macrophages and M2 macrophages play opposing roles in tumor development (19, 27). Recent studies have focused on CD163 staining in representative LARC tissues to investigate the clinical significance of TAMs (28, 29). Studies have shown that a higher density of M2 macrophages strongly predicts a poor response to nCRT and shorter OS and DFS in colorectal cancer (CRC) patients (28, 29). In our study, the number of TAMs/HPF was an independent predictor for the response to nCRT, as intense TAM staining was associated with a poor pathological response. Mechanistically, numerous cytokines and pathways, such as the IL-6R/STAT3/miR-204-5p pathway, were found to be involved in chemoresistance mediated by macrophages (30, 31). After radiotherapy, altered inflammatory cytokines (such as IL-6, IL-10, and TNF) lead to the recruitment of TAMs with a tissue reparative phenotype and contribute to tumor recurrence and metastasis (31). However, the studies above focused on the dynamics and effect of TAMs after radiation, and the predictive role of TAMs is poorly understood. Our present study showed that TAMs before nCRT play an important role in CRT resistance. Our results may partially be explained by the positive crosstalk between TAMs and cells related to immune suppression, including Th2 cells, cancer‐associated fibroblasts, Tregs, and MDSCs, and the negative crosstalk with CTLs (19, 32). This hypothesis needs to be further investigated in future studies.

A low number of Tregs was demonstrated to be strongly associated with pCR and better clinical outcomes in several cancers (9, 33, 34); therefore, it is shocking that no association between Tregs and clinical response was observed in the logistic regression analysis. The selection of truly representative target areas is a challenge common to all studies using biopsy samples and can be particularly difficult when using tissue from pretreated patients with significant variation in treatment response. For all studies using biopsy samples, whether the target is representative is a common problem (35). Although biopsies are carefully sampled by experienced endoscopists, it cannot be ruled out that the lack of a correlation between Treg density in tumors may be a problem associated with sampling. However, the number of Tregs was significantly different between the two groups via the Mann–Whitney U test, which proved the robustness of our study.

In this study, we observed that a high infiltration rate of Tregs was associated with a high infiltration rate of CTLs. Our findings could be explained by the fact that similar mechanisms are shared by CTLs and Tregs when infiltrating tumors (36, 37). For T cells to initiate extravasation, they must interact the CD44, CD62L or PSGL with endothelial selectin. All these molecules are expressed on the membranes of Tregs and activated CTLs. In addition, most of the chemokine receptors that mediate CD8+ T cell extravasation, such as CCR5, CXCR3 and CXCR6, are expressed on Tregs. As such, it is logical that Tregs and CTLs co-infiltrate a tumor. A positive correlation between TAMs and Tregs was also found. As discussed above, TAMs can directly recruit Tregs and activate them to establish the immunosuppressive TME (19). Numerous chemokines, such as CCL20 and CCL22, have been reported to participate in this process (38).

Regarding clinicopathological features, a higher density of TAMs was correlated with a positive lymph node stage in our study. As the major component of TIICs, TAMs were well-known to promote distant metastasis by multiple mechanisms (39, 40), and current studies found that the tumor cells present in lymph nodes maybe an important source of systemic metastasis (41, 42). In the most recent study by Chen et al, TAMs were found to significantly higher in breast primary tumor with lymph node metastasis compared with tumor tissues without lymph node involvement (43).

As the discussion above, the TAMs was found to be associated with nodal stage in present study, but no statistical relationship was found between the TAMs and tumor size, these result aroused our interest in exploring the mechanism underlying the role of M2 TAMs in lymph node metastasis in the early T stage.

This study had some limitations: first, this was a relatively small cohort of patients; thus, some clinically relevant factors, such as cT classification, failed to show significant differences. Therefore, these results must be confirmed in larger samples and through multicenter studies. Second, the clinicopathological parameters included are not comprehensive enough, which may lead to inefficiency of the model. In addition, the analyses performed were only correlation analyses, and further experimental studies are needed to explore the potential mechanism of these correlations.

Based on our study, these positive immunoregulatory features in preoperative evaluation may be suggestive for the need of combining current neoadjuvant regimens with an immune-modulating therapy to achieve better therapeutic outcome and survival in cancer treatment. However, as a correlation analyses with a relatively small cohort of patients, it still a long way to transform the results of current study into clinical applications.

In conclusion, this study proved that Tregs in tumor tissues were positively correlated with TAMs and CTLs. The density of tumor-infiltrating TAMs is a significant factor for lymph node metastasis. These findings suggest that TAMs and CTLs are predictive markers for the sensitivity of nCRT and might aid in clinical decision-making regarding the delivery of improved therapies for LARC. We believe that our data will provide the foundation for developing new prognostic biomarkers and improving the nCRT treatment outcomes of LARC patients. Besides, as total neoadjuvant therapy(TNT) with higher rate of achieving pCR emerging as an alternative treatment to LARC, the predictive role of CTLs, TAMs and Trgs play in LARC patients received TNT aroused our great interest and will be the next focus.

The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by Ethics Committee of the Fifth affiliated Hospital of Sun Yat-sen University and Harbin Medical University Cancer Hospital. The patients/participants provided their written informed consent to participate in this study.

HZ, BZ and QZ conceived and designed the experiments. YY, WT, LQS, PL, XG, and LS conducted and analyzed the data. YY and WT wrote this manuscript. All authors contributed to the article and approved the submitted version.

This study was financially supported from Guangdong Basic and Applied Basic Research Foundation (Grant Number: 2021A1515010421), National Natural Science Foundation of China (Grant Number. 31571417) and Natural Science Foundation of Zhejiang Province (Grant Number. LZ21H160006).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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