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Simple Summary: Immune checkpoint inhibitors (ICIs) have made an indelible mark on metastatic colorectal cancer patients with microsatellite instability-high (MSI-H) instead of microsatellite stability (MSS) tumors. Clinical studies have explored the introduction of ICIs into treatment for locally advanced rectal cancer patients with MSS; however, the outcomes are still limited. Different studies have provided valuable information for understanding the expression and clinical significance of PD-1 and PD-L1 during neoadjuvant chemoradiotherapy (nCRT). However, the value of CTLA-4 and CD86 after nCRT has not been fully studied. We found that CD86 expression was significantly lower in the nCRT-treated group rather than CTLA-4. Improved overall survival in patients with lower CD86 expression could only be observed in patients with low rather than high t/sCD8+ cell densities. Our findings reveal the relationship between the immunosuppressive microenvironment and the CTLA-4/CD86 pathway after nCRT. The synergistic effect of combining immune checkpoint inhibitors (ICIs) with neoadjuvant chemo(radio)therapy (nCRT) in colorectal cancer is still limited. We aimed to understand the impact of nCRT on the tumor microenvironment and to explore favorable immune markers of this combination. Herein, we investigated the expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), CD86, CD4, and CD8 after nCRT and its association with clinicopathological characteristics. Immunostaining of immune-related molecules was performed in 255 surgically resected specimens from rectal cancer patients treated with nCRT. CD4 and CD8 expression on the tumor (tCD4/CD8), stroma (sCD4/CD8), and invasive front (iCD4/CD8) was evaluated. The expression levels of immune-related molecules were significantly lower in the nCRT-treated group, except for CTLA-4 and sCD8. However, patients with higher sCD8+ cell density and CTLA-4 expression had better progression-free survival (PFS) and distant metastasis-free survival (DMFS). In addition, higher CD86 expression was associated with poorer overall survival (OS). Higher CTLA-4 expression was associated with higher tCD8+ cell density, whereas CD86 expression was correlated with the cell density of t/sCD8. Prognostic analysis confirmed that the relationships between CTLA-4 and DMFS as well as CD86 and OS were significantly correlated in low rather than high CD8+ cell density. Further the combination of CD8+ cell density and CD86 expression was shown to be an independent prognostic factor of OS, whereas the combination of CTLA-4 was not for DMFS. Together, these results demonstrate significant correlations between CD86 expression and t/sCD8+ cell density in rectal cancer after nCRT and could potentially have clinical implications for combining ICIs and nCRT. [ABSTRACT FROM AUTHOR]

Radioresistance is one of the main causes of cancer treatment failure, which leads to relapse and inferior survival outcome of cancer patients. Liquid-liquid phase separation (LLPS) of proteins is known to be involved in various biological processes, whereas its role in the regulation of radiosensitivity remains largely unknown. In this study, we characterized NONO, an RNA/DNA binding protein with LLPS capacity, as an essential regulator of tumor radioresistance. In vitro assay showed that NONO involved in DNA repair via non-homologous end joining (NHEJ) manner. NONO knockout significantly reduced DNA damage repair and sensitized tumor cells to irradiation in vitro and in vivo. NONO overexpression was correlated with an inferior survival outcome in cancer patients. Mechanically, NONO was associated with nuclear EGFR (nEGFR). Both irradiation and EGF treatment induced nEGFR accumulation, thereby increased the association between NONO and nEGFR. However, NONO was not a substrate of EGFR kinase. Furthermore, NONO promoted DNA damage-induced DNA-PK phosphorylation at T2609 by enhancing the interaction between EGFR and DNA-PK. Importantly, NONO protein formed high concentration LLPS droplets in vitro, and recruited EGFR and DNA-PK. Disruption of NONO droplets with LLPS inhibitor significantly reduced the interaction between EGFR and DNA-PK, and suppressed DNA damage-induced phosphorylation of T2609-DNA-PK. Taken together, LLPS of NONO recruits nuclear EGFR and DNA-PK and enhances their interaction, further increases DNA damage-activated pT2609-DNA-PK and promotes NHEJ-mediated DNA repair, finally leads to tumor radioresistance. NONO phase separation-mediated radioresistance may serve as a novel molecular target to sensitize tumor cell to radiotherapy., Competing Interests: None., (AJCR Copyright © 2021.)

Siglec15 is a recently characterized immunosuppressive transmembrane protein, which expresses in various types of solid tumors and promotes cancer development. Several studies reported that Siglec15 is a prognostic biomarker of cancer patients, and targeting Siglec15 may be a promising strategy for cancer therapy. However, the regulation of Siglec15 function remains unclear. Here we show that the immunosuppression activity of Siglec15 is largely modulated by N-glycosylation. Through mass spectrum and site mutation analysis, we identified that Siglec15 was extensively glycosylated at N172 (N173 for mouse) in cancer cells. Meanwhile, Siglec15 N172Q had a similar molecular weight with PNGase-F-treated Siglec15, suggesting N172 as the only one glycosylation residue. In xenograft model, glycosylation deficiency of Siglec15 reduced tumor growth in C57BL/6 mice, but had no impact in nude mice, indicating the requirement of N-glycosylation for immunosuppressive function of Siglec15. Furthermore, colorectal cancer patients with high Siglec15 expression had a poor response to neoadjuvant chemo-radiotherapy and short survival time. Interestingly, removal of N-glycosylation enhances the detection of Siglec15, which may be employed in the prediction of immunotherapy response. Together, our results disclose a pivotal role of glycosylated Siglec15 in tumor immune escape, which may be a therapeutic target for cancer immunotherapy., Competing Interests: None., (AJCR Copyright © 2021.)

Tissue-derived RNA, DNA and protein samples become more and more crucial for molecular detection in clinical research, personalized and targeted cancer therapy. This study evaluated how to biobanking colorectal tissues through examining the influences of cold ischemic time and freeze-thaw cycles on RNA, DNA and protein integrity. Here, 144 pairs of tumor and normal colorectal tissues were used to investigate the impact of cold ischemic times (0-48h) on RNA, DNA and protein integrity at on ice or room temperature conditions. Additionally, 45 pairs of tissues experienced 0-9 freeze-thaw cycles, and then the RNA, DNA and protein quality were analyzed. On ice, RNA, DNA and protein from colorectal tumor and normal tissues were all stable up to 48h after surgery. At room temperature, RNA in colorectal tumor and normal tissues began to degrade at 8h and 24h, respectively. Meanwhile, the tumor tissues DNA degradation occurred at 24h after surgery at room temperature. Similarly, the protein expression level of tumor and normal tissues began to change at 24h after the surgery at room temperature. Interestingly, tissue RNA and DNA remained stable even after 9 freeze-thaw cycles, whereas the proteins levels were remarkably changed after 7 freeze-thaw cycles. This study provided a useful evidence on how to store human colorectal tissues for biobanking. Preserving the surgical colorectal tissue on ice was an effective way to prevent RNA, DNA and protein degradation. Importantly, more than 7 repeated freeze-thaw cycles were not recommended for colorectal tissues., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)