Colorectal cancer (CRC) is the third most common and the fourth leading cause of cancer-related deaths worldwide (1). Rectal cancer accounts for approximately 30% of CRC cases, with locally advanced rectal cancer (LARC) the most widespread form of rectal cancer (2). The standard treatment for LARC is neoadjuvant chemoradiation (nCRT), followed by total mesorectal excision. However, LARC’s sensitivity to nCRT varies among patients, with only 4%-20% of patients often achieving pathological complete response (pCR) after surgery (3).

Immune checkpoint inhibitors (ICIs), such as programmed cell death-1 inhibitor (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitor, have dramatically changed the treatment landscape of solid tumors recently. A high density of tumor-infiltrating lymphocytes (TILs), particularly CD8+ T lymphocytes, in rectal cancer is associated with better prognosis, suggesting that ICIs have promising antitumor effects in rectal cancer (4, 5).Radiation enhances CD8+ T cell infiltration in tumors and improves local tumor control, long-term survival, and protection against tumor rechallenge (6–8). The NICHE study (ClinicalTrials.gov: NCT03026140) showed that neoadjuvant treatment with ipilimumab plus nivolumab resulted in major pathological responses in 100% of mismatch repair (MMR)-deficient colon cancer cases without compromising surgery (9). There are ongoing clinical trials (NCT 03127007, NCT03102047, NCT02948348) evaluating the effectiveness of combining chemoradiation with ICIs to treat rectal cancer.

Reportedly, the soluble form of PD-L1 plays a role in immune suppression and is closely linked to poor prognosis in solids tumors (10–12). A study evaluating plasma soluble PD-1/PD-L1 levels in 117 advanced rectal cancer patients treated with nCRT revealed that high sPD-L1 levels after chemoradiation were associated with worse disease-free survival (13). However, no reports to date have examined the significance of the soluble form of co-stimulatory molecules, such as CD28-CD80/CD86, GITR-GITRL, CD27, and ICOS, in LARC patients treated with nCRT.

This study prospectively investigated the clinical implications of the changes in the soluble forms of PD-L1, CD80, CD86, CD28, GITR, GITRL, CD27, and ICOS in LARC patients treated with nCRT.

This study, to our knowledge, is the first prospective study to examined the significance of sPD-L1, sCD80, sCD86, sCD28, sGITR, sGITRL, sCD27, and sICOS in LARC patients treated with chemoradiotherapy. Our results showed that these immune checkpoint molecules correlate strongly with each other in LARC patients. This correlation may suggest a common source or regulation of these soluble proteins. The plasma levels of the 8 soluble immune checkpoint molecules decreaded during nCRT and were restored after nCRT, especially in responders to nCRT. The variation patterns differed significantly between the two groups, with more pattern 2 changes in good responders than in poor responders and more pattern 3 changes in poor responders than in good responders. Although single sICMs showed some prognostic relevance in the ROC curve and univariate logistic regression analyses, none of them was a good predictor of treatment response. To improve predictive performances, three scores incorporating these eight sICMs were constructed respectively at three time-points. The Pre-Model exhibited a higher predictive value than single sICMs and was identified as an independent predictor of treatment response.

In the immune system, immune checkpoints can be divided into co-inhibitory molecules, such as PD-1/PD-L1, and co-stimulatory molecules, such as CD80/CD86, ICOS/ICOS-L. The soluble form of immune checkpoint molecules is typically generated by the proteolytic cleavage of the membrane-bound form of the proteins, such as PD-L1 (14), or by the translation of alternatively spliced mRNA, such as CD80 (15) and CD86 (16).

The study described for the first time the changes of soluble immune checkpoint molecules (sPD-L1, sCD80, sCD86, sCD28, sGITR, sGITRL, sCD27, and sICOS) in patients with LARC following radiotherapy. The changes showed significant differences between responders and poor responders after nCRT. Good responders have more patients with decreased sICMs during CRT than poor responders; poor responders have more patients with increased sICMs during nCRT. These results suggest that chemoradiotherapy could remodel peripheral immune components. Additionally, one further points merit mention. Stereotactic body radiotherapy (SBRT) is more immunogenic than conventional fractionated radiotherapy (17). A recent study revealed that the two different radiotherapy fractioned regimens have a different effect on sPD-L1 changes; the sPD-L1 level continuously increased in the SBRT group but decreased after 1-month in the conventional fractionated radiotherapy group (18). However, the explanation for these phenomena is absent.

The sPD-1/sPD-L1 is the most widely studied soluble immune checkpoint molecule. Membrane-bound forms of PD-1 (mPD-L1) expressed on both tumor and immune cells, which may be the most prominent sources of sPD-L1 (14, 19). Preclinical studies have shown that radiotherapy increases the expression of PD-L1 in tumor lesions (6, 20). Hecht et al. examined PD-L1 expression in 63 pre-nCRT cases and matched them with post-nCRT sample expression to show that PD-L1 was upregulated in rectal cancer patients after nCRT (21). However, our results showed that sPD-L1 levels did not significantly increase after nCRT, which is inconsistent with its elevation after nCRT in published literature (13). Since our follow-up period was not long enough, further studies with a larger number of patients are required to clarify this contradictory point. In addition to changes of the sPD-L1 levels following radiotherapy, whether sPD-L1 can bind to PD-1 and deliver an inhibitory signal is debatable. Several studies have shown that elevated sPD-L1 is associated with poor prognosis in a wide variety of cancers, including malignant melanoma (22), lung cancer (23), hepatocellular carcinoma (24), multiple myeloma (25), and renal cell carcinoma (26). The study by Hyun Ju et al. firstly demonstrated that patients with a high level of initial sPD-L1 had a poor survival after RT in HCC patients (18). Similarly, our study found that patients with a high level of initial sPD-L1 trends to have poor response to chemoradiotherapy, although not statistically significant ( Figure 5 ). These findings suggest that sPD-L1 might represent a biomarker for predicting the treatment response to chemoradiotherapy in LARC patients.

CD80/CD86-CD28 and ICOS-ICOSL belong to the B7-CD28 superfamily and provide important co-stimulatory signals to augment and sustain a T-cell response (27). GITR-GITRL and CD27 are important members of the TNF superfamily and also export co-stimulatory signals to support CD8 T-cell differentiation, proliferation, and NK cell functions (28). However, these soluble co-stimulatory molecules predominantly associate with poor prognosis in solid tumor patients (29–32), which is not consistent with the role of their corresponding membrane-bound forms in T cell activation. In our study, our result also support this phenomenon ( Figure 5 ). One possible assumption for their association with poor prognosis is that their binding to corresponding immune checkpoint molecules in immune cells may prevent membrane receptor-ligand binding, thereby counteracting the co-stimulatory signals of T cells. Although the exact mechanism remains unclear, the soluble form of immune checkpoint molecules is demonstrated to involve in tumor immunity and affects the prognosis of patients.

However, none of the sICMs in our study was a reliable predictive marker. This, in fact, is not surprising. A single immune checkpoint molecule may not be powerful enough to predict treatment response. Multi-marker analyses that incorporate single markers into a panel may have more promising clinical application prospects. We also found that the Pre-model, incorporated baseline sICMs was an independent prognostic factor. Thus, there is promise for the development of a noninvasive blood-based model to serve as more convenient biomarker for the prediction of treatment response.

There are several limitations to our study. First, this study was a single-center exploratory analysis, and the sample size was limited. Second, chemotherapy might have influenced the levels of sICMs in our patients, although little is currently known about this possibility. Third, we did not validate our findings with another independent external study.

In conclusion, chemoradiotherapy significantly influenced the plasma levels of sPD-L1, sCD80, sCD86, sCD28, sCD27, and sICOS in LARC patients, and their changes differed markedly between good responders and poor responders. Patients with high baseline levels of these molecules tended to respond poorly to nCRT. A combined diagnostic panel that incorporates them had an obvious advantage over the corresponding single molecules. These findings suggest soluble immune checkpoint molecules have clinical values that may aid the future development of radiotherapy and immunotherapy.

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

The studies involving human participants were reviewed and approved by the Research Ethics Board of Shandong Cancer Hospital. The patients/participants provided their written informed consent to participate in this study.

All authors contributed to the study conception and design. Data collection/preprocessing and analysis were performed by CL and PW. CL and PW contributed equally to the first draft of the manuscript, and all the listed authors revised the submitted manuscript and approved its final version before submission.

This work was supported by the following grants: National Natural Science Foundation of China (Grant No. 81871895) and Young Taishan Scholars and Academic Promotion Program of Shandong First Medical University (Grant No. 2019RC003).

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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