膀胱癌為全球十大惡性癌症之一。常見的膀胱癌治療方法包括卡介苗疫苗療法、手術切除、化學治療、放射線治療和免疫檢查點抑制劑療法。早期膀胱癌病患在接受治療後，存活率大多會有顯著改善，但對於晚期膀胱癌病患目前並無普遍有效的療法。先前臨床資料指出部分晚期的膀胱癌病患會有neutrophil異常增生的現象。其它癌症的研究則表明neutrophil會參與腫瘤的進展，但neutrophil在膀胱癌中的作用仍未明瞭。我們的研究闡述了腫瘤微環境中癌細胞與免疫細胞的交互作用如何促進膀胱癌的進展。
已知CSF3會促進neutrophil的增生且CSF3R為CSF3的受體。我們發現膀胱癌細胞5637有CSF3表現卻沒有 CSF3R表現。相對地，骨髓衍生細胞HL60則有CSF3R表現而沒有CSF3表現。收集5637細胞的conditioned medium 去培養HL60細胞會使其大量增生。而在5637細胞中降低CSF3的表現並不會影響細胞本身的生長情況，但會抑制5637細胞 conditioned medium增加HL60細胞增生的效果，說明膀胱癌細胞所表現的CSF3不會直接影響其本身的生長而是去促進neutrophil的增生。另一方面，我們也發現HL60細胞所表現的ELANE可以提升5637細胞在低濃度血清下的存活能力。除此之外，更進一步的研究結果表明5637細胞所分泌的CSF3可以提升HL60細胞的浸潤程度。綜合上述的研究結果，我們推測膀胱癌細胞會分泌CSF3去提高neutrophil的增生與浸潤程度再藉由neutrophil所分泌的ELANE來增加其本身的存活能力。
整體而言，在本研究中我們提供了新的觀點，清楚地說明膀胱癌細胞在腫瘤微環境中是如何誘導neutrophil來協助自身的生長。另外使用來自the cancer genome atlas (TCGA) 資料去做膀胱癌的預後分析，再次表明CSF3和ELANE之間的關係對於pro-tumor膀胱癌腫瘤微環境的形成佔有重要地位，2者之間除了有正相關表現外，ELANE表現量越高的病患會有越低的overall survival。我們認為ELANE有潛力成為膀胱癌病患新的預後指標和治療標靶。

Granulocyte colony stimulating factor (G-CSF), encoded by CSF3, and its receptor G-CSFR, encoded by CSF3R, regulates neutrophil proliferation. However, the roles of CSF3 and CSF3R in bladder cancer progression remain elusive. Here, we report that CSF3 is highly expressed in a subgroup of bladder tumors, which display high neutrophil infiltration and low tumor purity. We observed that CSF3 was highly expressed in a subgroup of bladder tumors, which display high neutrophil infiltration and low tumor purity. We observed that CSF3 was highly expressed in T24 cells and basal-like bladder cancer 5637 cells but not in luminal-like bladder cancer RT4 cells. In contrast, CSF3R was highly expressed in myeloid-derived HL60 cells but not in 5637, T2 and RT4 bladder cancer cells. Knockdown of CSF3 did not affect growth of 5637 bladder cancer cells. However, the conditioned medium from 5637 bladder cancer promoted cell cycle of HL60 promyelocytic cells, and silencing CSF3 in 5637 cells attenuated this effect. The clonogenic assays showed that the conditioned medium from HL60 promyelocytic cells rendered 5637 bladder cancer cells resistant to low serum stress, whereas knockdown of ELANE in HL60 cells diminished this effect. Kaplan-Meier survival analysis showed that high ELANE expression was associated with poor survival in bladder cancer, and bladder tumors harboring ELANE-high/CSF3R-high signature displayed worse overall survival outcomes. Our findings provide insights into the roles of CSF3 and CSF3R in bladder cancer cells and myeloid-derived cells for their potential crosstalk in tumor progression.

摘要.............................................................1
ABSTRACT.........................................................3
ACKNOWLEDGEMENT..................................................4
INTRODUCTION.....................................................8
Bladder cancer...................................................8
G-CSF and G-CSFR................................................10
Tumor microenvironment..........................................11
Neutrophils and myeloid-derived suppressor cells................12
Neutrophil elastase.............................................14
TGF-β...........................................................15
Aims of study...................................................16
MATERIALS AND METHODS...........................................17
Cell lines and cell culture.....................................17
Reagents........................................................17
Quantitative real-time polymerase chain reaction (qRT-PCR)......17
Plasmids........................................................18
Lentiviral transfection.........................................18
Clonogenic assay................................................18
Cell cycle assay................................................18
Cell apoptosis assay............................................19
Enzyme-linked immunosorbent assay (ELISA).......................19
Cell Matrigel infiltration assay................................19
Xenograft model and neutrophil to lymphocyte ratio assay........20
Public domain data and statistical analysis.....................20
RESULTS.........................................................21
Distinct CSF3 and CSF3R expression in bladder cancer and myeloid-derived cells...................................................21
Loss of CSF3R expression in differentiated myeloid-derived cells ................................................................21
Bladder cancer cells secrete factors to enhance proliferation of myeloid-derived cells...........................................22
Bladder cancer cells secret CSF3 to enhance proliferation of myeloid-derived cells...........................................22
Myeloid-derived cells secret factors to enhance proliferation of bladder cancer cells............................................22
ELANE is associated with poor prognosis of bladder cancer.......23
Myeloid-derived cells secret ELANE to enhance growth of bladder cancer cells....................................................24
High CSF3 expression is associated with neutrophil infiltration in bladder tumors..................................................24
TGF-β expression in the bladder cancer..........................25
DISCUSSION......................................................26
FIGURES.........................................................29
Figure 1. CSF3 and CSF3R expression in bladder cancer and myeloid-derived cells...................................................29
Figure 2. CSF3R expression in undifferentiated myeloid-derived cells...........................................................30
Figure 3. Effect of conditioned medium from bladder cancer cells on proliferation of myeloid-derived cells.......................31
Figure 4. CSF3 secretion from bladder cancer cells promotes proliferation of myeloid-derived cells..........................32
Figure 5. ELANE expression in myeloid-derived cells.............34
Figure 6. ELANE expression is associated with poor prognosis of bladder cancer patients.........................................35
Figure 7. The effect of ELANE from myeloid-derived cells on proliferation of bladder cancer cells...........................37
Figure 8. CSF3 and CSF3R expression is associated with neutrophil infiltration of bladder tumors..................................39
Figure 9. Elane expression in granulocyte-like myeloid derived suppressor cells................................................41
Figure 10. Model of the crosstalk between CSF3 secretion from bladder cancer cells and ELANE secretion from myeloid-derived cells in bladder tumor microenvironment.........................42
TABLES..........................................................43
Table 1. shRNA clones...........................................43
Table 2. qRT-PCR primers and probes.............................44
Table 3. Public domain datasets.................................45
REFERENCES......................................................46

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