Background

The interplay between cancer cells and stromal components, including soluble mediators released from cancer cells, contributes to the progression of pancreatic ductal adenocarcinoma (PDAC). Here, we set out to identify key secreted proteins involved in PDAC progression.

Methods

We performed secretome analyses of culture media of mouse pancreatic intraepithelial neoplasia (PanIN) and PDAC cells using Stable Isotope Labeling by Amino acid in Cell culture (SILAC) with click chemistry and liquid chromatography-mass spectrometry (LC-MS/MS). The results obtained were verified in primary PDAC tissue samples and cell line models.

Results

Complement factor B (CFB) was identified as one of the robustly upregulated proteins, and found to exhibit elevated expression in PDAC cells compared to PanIN cells. Endogenous CFB knockdown by a specific siRNA dramatically decreased the proliferation of PDAC cells, PANC-1 and MIA PaCa-II. CFB knockdown induced increases in the number of senescence-associated-β-galactosidase (SA-β-gal) positive cells exhibiting p21 expression upregulation, which promotes cellular senescence with cyclinD1 accumulation. Furthermore, CFB knockdown facilitated downregulation of proliferating cell nuclear antigen and led to cell cycle arrest in the G1 phase in PDAC cells. Using immunohistochemistry, we found that high stromal CFB expression was associated with unfavorable clinical outcomes with hematogenous dissemination after surgery in human PDAC patients. Despite the presence of enriched CD8⁺ tumor infiltrating lymphocytes in the PDAC tumor microenvironments, patients with a high stromal CFB expression exhibited a significantly poorer prognosis compared to those with a low stromal CFB expression. Immunofluorescence staining revealed a correlation between stromal CFB expression in the tumor microenvironment and an enrichment of immunosuppressive regulatory T-cells (Tregs), myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). We also found that high stromal CFB expression showed a positive correlation with high CD8⁺/Foxp3⁺ Tregs populations in PDAC tissues.

Conclusions

Our data indicate that CFB, a key secreted protein, promotes proliferation by preventing cellular senescence and is associated with immunological tumor promotion in PDAC. These findings suggest that CFB may be a potential target for the treatment of PDAC.

中文翻译：

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补体因子 B 调节细胞衰老并与胰腺癌不良预后相关

 背景

癌细胞和基质成分（包括癌细胞释放的可溶性介质）之间的相互作用，有助于胰腺导管腺癌（PDAC）的进展。在这里，我们着手鉴定参与 PDAC 进展的关键分泌蛋白。

 方法

我们使用细胞培养中氨基酸稳定同位素标记 (SILAC)、点击化学和液相色谱-质谱 (LC-MS/MS) 对小鼠胰腺上皮内瘤变 (PanIN) 和 PDAC 细胞的培养基进行分泌组分析。获得的结果在原代 PDAC 组织样本和细胞系模型中得到了验证。

 结果

补体因子 B (CFB) 被确定为强烈上调的蛋白质之一，并且发现与 PanIN 细胞相比，PDAC 细胞中的表达升高。通过特定 siRNA 敲低内源性 CFB，可显着降低 PDAC 细胞、PANC-1 和 MIA PaCa-II 的增殖。 CFB敲低诱导衰老相关β-半乳糖苷酶（SA-β-gal）阳性细胞数量增加，表现出p21表达上调，从而通过cyclinD1积累促进细胞衰老。此外，CFB 敲低促进了增殖细胞核抗原的下调，并导致 PDAC 细胞的细胞周期停滞在 G1 期。使用免疫组织化学，我们发现高基质 CFB 表达与人类 PDAC 患者术后血行播散的不良临床结果相关。尽管 PDAC 肿瘤微环境中存在富集的 CD8 ⁺肿瘤浸润淋巴细胞，但与基质 CFB 低表达的患者相比，基质 CFB 高表达的患者预后明显较差。免疫荧光染色揭示了肿瘤微环境中基质 CFB 表达与免疫抑制调节 T 细胞 (Treg)、骨髓源性抑制细胞 (MDSC) 和肿瘤相关巨噬细胞 (TAM) 富集之间的相关性。我们还发现，高基质 CFB 表达与 PDAC 组织中高 CD8 ⁺ /Foxp3 ⁺ Tregs 群体呈正相关。

 结论

我们的数据表明，CFB 是一种关键的分泌蛋白，通过防止细胞衰老来促进增殖，并与 PDAC 中的免疫肿瘤促进相关。这些发现表明 CFB 可能是治疗 PDAC 的潜在靶点。