Glioblastoma (GBM) is the most common primary malignant tumor in the central nervous system (CNS) and exhibits a poor prognosis (1). GBM is therapeutically intractable and refractory to currently available multimodal treatments including surgery, radiotherapy, and chemotherapy (2). Most newly diagnosed GBMs present as a unifocal GBM (uGBM), whereas GBMs with multiple lesions are relatively rare, as the latter account for only 0.5%–20% of all GBMs based on histopathological or radiological diagnosis (3–9). Multiple GBMs are categorized into multifocal and multicentric GBMs (mGBMs). The foci of multifocal GBMs are close to each other, suggesting a physical connection; in contrast, the foci of multicentric are located in different lobes or hemispheres without any obvious dissemination route (10, 11). Nevertheless, prior publications have shown no significant utility in the distinction between multifocal and multicentric GBMs (5, 7, 10–13). Therefore, per the convention of previous studies (7, 10, 11), we use the term “mGBM” in the present study to represent our cases with multiple GBM lesions within a single patient. According to previous reports, mGBMs are associated with even worse clinical outcome and poorer survival times compared to uGBMs (7, 10, 14, 15). However, the pathogenic mechanisms and clinical characteristics of mGBMs are still largely unclear, and standards of care for mGBMs are not well defined. Therefore, new treatment strategies, including immunotherapies, need to be developed for mGBMs.

The tumor microenvironment (TME) is a critical regulator of pathogenesis and therapeutic responses in GBMs (16). CD8⁺ tumor-infiltrating lymphocytes (TILs) have been demonstrated to be important components of the TME and predict improved survival in several human malignancies, including breast cancer (17), ovarian cancer (18), and colorectal cancer (19). CD8⁺ TILs have also been detected in the TME of GBMs; however, the prognostic effect of CD8⁺ TILs in GBMs is still controversial (20–23). Moreover, the role of CD8⁺ TILs in mGBMs has not yet been elucidated. Thus, in the present study, we investigated the infiltration of CD8⁺ lymphocytes in a series of 57 newly diagnosed mGBMs and assessed the effects of CD8⁺ TILs and other clinical parameters on the prognosis of mGBM patients. Taken together, our findings might shed light on immuno-microenvironmental mechanisms of mGBM biology.

Although GBMs have been extensively investigated, few studies have focused on multifocal and multicentric GBMs (mGBMs) possibly due to their rare occurrences. Since the majority of newly diagnosed GBMs are unifocal, mGBMs may have distinct molecular mechanisms, microenvironmental characteristics, and clinical courses (10, 11), which have not been clearly elucidated. Moreover, current standard radio-chemotherapy shows inferior therapeutic effects in mGBMs compared with those in uGBMs (15). Therefore, the development of novel mGBM-targeted treatment strategies is clinically relevant.

Previous studies have demonstrated that the immunosuppressive TME plays an important role in the development and progression of GBMs (24, 25). A hallmark of GBM-induced immunosuppression is the inhibition of CD8⁺ effector T-cell mediated anti-tumor responses (26, 27). A number of GBM-derived factors trigger reprogramming of immune cells and inhibit the accumulation and activation of cytotoxic CD8⁺ TILs (24, 28). Although some studies have failed to show a relation between CD8⁺ TILs and clinical outcomes (20, 21), several studies have observed a significant correlation between decreased CD8⁺ TILs and poorer patient survival in GBMs (22, 23, 29). In addition, previous studies did not investigate mGBMs and uGBMs separately. Therefore, in the present study, we specifically examined the CD8⁺ TILs in mGBMs. We found that mGBMs were associated with decreased CD8⁺ lymphocytic infiltration compared with uGBMs. Moreover, lower CD8⁺ TIL levels predicted shorter survival times in mGBM patients. Decreased CD8⁺ TIL levels indicate a more serious local immunosuppressive TME, which may facilitate the development of multifocality and promote the immune evasion of tumor cells. Therefore, inhibition of CD8⁺ lymphocytic infiltration might provide an immuno-microenvironmental basis for the formation and progression of multiple lesions of mGBMs. Moreover, our present results showed that CD8⁺ TIL levels were associated with preoperative KPS in mGBM patients, which suggests an interaction of systemic status and the local immune TME. Nevertheless, further studies are required to better understand the intrinsic mechanisms of this immunosuppression. Our present results also indicate that the immunomodulatory mechanism employed by mGBMs poses a considerable challenge to immunotherapy, since effective immunotherapy often requires the participation of functionally active CD8⁺ T cells (28, 30). Thus, counteracting the impairment of CD8⁺ TILs and promoting a sustained tumor-cell-directed cytotoxic T-cell response are necessary to overcome such immunosuppression and to establish efficacious immunotherapeutic treatments in mGBM patients.

Currently, specific clinical guidelines for the standard treatment of mGBMs are still unavailable (1). Unfortunately, mGBMs are treated in the same way as uGBMs, although they are biologically and clinically different from each other (5, 10, 11). Therefore, the efficacies of surgical treatment and radio-chemotherapy should be re-evaluated in mGBM patients. For example, the role of surgery for uGBMs has been well demonstrated by previous studies, and GTR significantly improves OS (31, 32). However, the optimal surgical management of mGBMs remains controversial (5). Some authors support stereotactic biopsy as the first choice, showing that radical resection increases postoperative morbidity without survival benefits (33–35); while other authors propose aggressive resection, reporting that cytoreductive surgery strongly influences OS in mGBM patients (6, 36, 37). In addition, the prognostic value of radiation therapy has not been clarified in mGBM patients, and the efficacies of whole-brain radiotherapy and 3D conformal radiotherapy are still under debate (13, 38, 39). In the present study, we found that TMZ chemotherapy was associated with improved OS of patients with mGBMs, which is consistent with findings from previous reports (15, 38). Therefore, TMZ systemic therapy is effective and beneficial and should be a major component of forthcoming therapeutic strategies for mGBMs.

In this study, we demonstrated decreased CD8⁺ lymphocytic infiltration in mGBMs and potential prognostic significance of CD8⁺ TIL levels in mGBM patients. Our results suggest that the local immunosuppressive TME might affect the development and progression of mGBMs.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

This study was approved by the institutional review board of the First Hospital of China Medical University (AF-SOP-07-1.1-01).

RW, YS, TH, XW, and LK cooperated to complete the experiment. RW, TH, YJ, DZ, JY, and SH contributed to the collection and analysis of data. RW, YS, TH, XW, and YJ participated in drafting the text and figures. SH and LK designed the study and gave indispensable guidance in drafting the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the Liaoning Revitalization Talents Program (no. XLYC1807253) and the National Natural Science Foundation of China (nos. 81772653 and 81402045).

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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