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Abstract：
Pancreatic ductal adenocarcinoma (PDAC) poorly responds to antineoplastic agents. Discrepancies between preclinical success and clinical failure of compounds has been a continuous challenge and major obstacle in PDAC research.
To investigate the association of the tumor microenvironment (TME) composition and gemcitabine metabolizing enzyme (GME) expression in vitro and several in vivo models.
mRNA expression and protein levels of GME (cytosolic 5\'-nucleotidase 1 A; NT5C1A, cytidine deaminase; CDA, deoxycytidine kinase; DCK), gemcitabine transporters (ENT1, ENT2, RRM1, RRM2) and stromal components (hyaluroninc acid, podoplanin, masson trichrome, picrosirius) were assessed by qRT-PCR and immunohistochemistry in murine LSL-KrasG12D/+;LSL-Trp53R172 H/+; Pdx-1-Cre (KPC), orthotopically transplanted mice (OTM), human primary resected PDAC tissue (hPRT), corresponding patient-derived xenograft (PDX) mice, and KPC-SPARC-/- mice. mRNA expression of GME was analyzed in PDAC cell lines (Panc-1, MIA PaCa, BXPC3 and L3.6) upon incubation on collagen or pancreatic stellate cell (PSC) conditioned media by qRT-PCR.
Endogenous KPC tumors exhibited significantly higher levels of GME compared to OTM. However, GME levels did not differ between hPRT and corresponding PDX mice. Using Kendalls Tau correlation coefficient we did not show a significant correlation of GME and components of the TME except for NT5C1A and hyaluronic acid in PDX mice (p=0.029). GME were not significantly altered upon SPARC depletion in vivo, and upon treatment with PSC-conditioned media or incubation on collagen plated dishes in vitro.
Our findings suggest that the expression of GME is independent from the deposition of stromal components. KPC mice are most appropriate to study stromal composition whereas PDX mice maintain GME expression of the corresponding hPRT and could be best suited for pharmacokinetic studies.
To investigate the association of the tumor microenvironment (TME) composition and gemcitabine metabolizing enzyme (GME) expression in vitro and several in vivo models.
mRNA expression and protein levels of GME (cytosolic 5\'-nucleotidase 1 A; NT5C1A, cytidine deaminase; CDA, deoxycytidine kinase; DCK), gemcitabine transporters (ENT1, ENT2, RRM1, RRM2) and stromal components (hyaluroninc acid, podoplanin, masson trichrome, picrosirius) were assessed by qRT-PCR and immunohistochemistry in murine LSL-KrasG12D/+;LSL-Trp53R172 H/+; Pdx-1-Cre (KPC), orthotopically transplanted mice (OTM), human primary resected PDAC tissue (hPRT), corresponding patient-derived xenograft (PDX) mice, and KPC-SPARC-/- mice. mRNA expression of GME was analyzed in PDAC cell lines (Panc-1, MIA PaCa, BXPC3 and L3.6) upon incubation on collagen or pancreatic stellate cell (PSC) conditioned media by qRT-PCR.
Endogenous KPC tumors exhibited significantly higher levels of GME compared to OTM. However, GME levels did not differ between hPRT and corresponding PDX mice. Using Kendalls Tau correlation coefficient we did not show a significant correlation of GME and components of the TME except for NT5C1A and hyaluronic acid in PDX mice (p=0.029). GME were not significantly altered upon SPARC depletion in vivo, and upon treatment with PSC-conditioned media or incubation on collagen plated dishes in vitro.
Our findings suggest that the expression of GME is independent from the deposition of stromal components. KPC mice are most appropriate to study stromal composition whereas PDX mice maintain GME expression of the corresponding hPRT and could be best suited for pharmacokinetic studies.
摘要：
背景：胰腺导管腺癌（PDAC）对抗肿瘤药物的反应较差。化合物的临床前成功和临床失败之间的差异一直是PDAC研究中的持续挑战和主要障碍。
目的：研究肿瘤微环境（TME）组成与吉西他滨代谢酶（GME）表达的关系。
方法：GME的mRNA表达和蛋白质水平（胞质5'-核苷酸酶1A；NT5C1A，胞苷脱氨酶;CDA,脱氧胞苷激酶；DCK），吉西他滨转运体（ENT1，ENT2，RRM1，RRM2）和基质成分（透明质酸，podoplanin,马森三色,picrosirius）通过qRT-PCR和免疫组织化学在鼠LSL-KrasG12D/+中进行评估;LSL-Trp53R172H/+;Pdx-1-Cre（KPC），原位移植小鼠（OTM），人原发性切除的PDAC组织（hPRT），相应的患者源性异种移植（PDX）小鼠，和KPC-SPARC-/-小鼠。在PDAC细胞系中分析GME的mRNA表达（Panc-1，MIAPaCa，BXPC3和L3.6）通过qRT-PCR在胶原蛋白或胰腺星状细胞（PSC）条件培养基上孵育后。
结果：与OTM相比，内源性KPC肿瘤表现出明显更高的GME水平。然而,GME水平在hPRT和相应的PDX小鼠之间没有差异。使用KendallsTau相关系数，除了PDX小鼠中的NT5C1A和透明质酸之外，我们没有显示GME与TME成分的显着相关性（p=0.029）。GME在体内SPARC耗竭后没有显著改变，并且在用PSC条件培养基处理或在体外胶原铺板培养皿上孵育后。
结论：我们的发现表明GME的表达与基质成分的沉积无关。KPC小鼠最适于研究基质组成,而PDX小鼠维持相应hPRT的GME表达,并且可能最适于药代动力学研究。
目的：研究肿瘤微环境（TME）组成与吉西他滨代谢酶（GME）表达的关系。
方法：GME的mRNA表达和蛋白质水平（胞质5'-核苷酸酶1A；NT5C1A，胞苷脱氨酶;CDA,脱氧胞苷激酶；DCK），吉西他滨转运体（ENT1，ENT2，RRM1，RRM2）和基质成分（透明质酸，podoplanin,马森三色,picrosirius）通过qRT-PCR和免疫组织化学在鼠LSL-KrasG12D/+中进行评估;LSL-Trp53R172H/+;Pdx-1-Cre（KPC），原位移植小鼠（OTM），人原发性切除的PDAC组织（hPRT），相应的患者源性异种移植（PDX）小鼠，和KPC-SPARC-/-小鼠。在PDAC细胞系中分析GME的mRNA表达（Panc-1，MIAPaCa，BXPC3和L3.6）通过qRT-PCR在胶原蛋白或胰腺星状细胞（PSC）条件培养基上孵育后。
结果：与OTM相比，内源性KPC肿瘤表现出明显更高的GME水平。然而,GME水平在hPRT和相应的PDX小鼠之间没有差异。使用KendallsTau相关系数，除了PDX小鼠中的NT5C1A和透明质酸之外，我们没有显示GME与TME成分的显着相关性（p=0.029）。GME在体内SPARC耗竭后没有显著改变，并且在用PSC条件培养基处理或在体外胶原铺板培养皿上孵育后。
结论：我们的发现表明GME的表达与基质成分的沉积无关。KPC小鼠最适于研究基质组成,而PDX小鼠维持相应hPRT的GME表达,并且可能最适于药代动力学研究。
