br Lung cancer cells with a higher
Lung cancer NS-638 with a higher level of OCT4 are more resistant to drug treatments . Regarding cancer stemness, OCT4 and CD44 are both representative stemness markers for several cancers. In the present study, the expression of OCT4 was up-regulated and CD44 was down-
regulated in 1:9 co-spheroids. Interestingly, the decreased CD44 ex-pression was reported to correlate with liver metastasis, tumor diﬀer-entiation, and malignancy stage in pancreatic cancer .
The uniqueness of this model is the tight packing of stroma that serves as a barrier to limit drug penetration. The sensitivity of to Abraxane combined with GEM (∼25%) was higher in the 2D model compared to that of the 3D model (∼40%) in Fig. 5. This result sug-gested that the major mechanism for the chemotherapy resistance of the cell sphere may be the strong barrier eﬀect from PSCs that decreases the penetration of drug. In addition, the chemotherapy resistance genes ABCC1 and DARPP-32 were up-regulated after MIA cells and PSCs were merged into spheroids. The ABCC1 transporter, highly expressed in cancer stem cells and linked to chemo-resistance and tumor recurrence , was up-regulated in the MIA-PSC spheroids. Another drug re-sistant marker DARPP-32, which is a phosphatase and kinase inhibitor associated with resistance against trastuzumab in breast cancer cells , was also up-regulated in the current spheroids. These results suggested that the ABCC1 gene might play another critical role in the eﬄux of drugs from the PSC cytoplasm.
PDX (patient-derived xenograft) animal model has better potential mimicking the characteristics of mirroring genetics, tumor hetero-geneity, and cancer microenvironment on the primary tumor in clinical patients. However, despite all of these advantages, the PDX animal model has several drawbacks. Firstly, the engraftment rate for PDX is low (usually under 20% for subcutaneous grafted primary tumor). Secondly, development of PDX usually takes 4–8 months, which will be a limiting factor for patients with rapidly progressive neoplasms like pancreatic cancer. Thirdly, the replacement of human by murine stroma will aﬀect the potency of anti-stromal drugs. In contrast, our 3D culture of pancreatic cancer and stellate cells on CS-HA plates most likely mi-mics the microenvironment of pancreatic cancer model and provide a faster, high-throughput screening of anti-cancer drugs.
The zebrafish model was considered as a translation model before the mice model. For observation of PDAC tumor metastasis in the mice model, fluorescent tumor cells need to be generated and injected into the spleen. These processes are time-consuming and complicated. Indeed, the mice model is more powerful than the zebrafish xenograft. However, it is impossible to evaluate the motility of transplanted cells in the mice model. We used the zebrafish model in this study as a proof-of-concept investigation for the co-culture spheroids that mimicked the pancreatic tumor tightly packed by the stroma. The cell line was not the best choice to study the characters of cancers in vivo because the characteristics of cell line may change after long time culture. The PDX (patient-derived xenograft) animal model could even better mimic the characteristics that exhibit on clinical patients, while modeling with several commercialized cell lines seems too coarse for personalized medication guidance. In the future, we will use the mice model for further investigation of the multiple cell type co-culture spheroid system. The tumorigenicity of pancreatic cancer cells has been de-monstrated recently by two diﬀerent zebrafish xenograft implantation models [62,63]. By yolk sac implantation, the pancreatic cancer cells displayed obvious metastatic behavior, and the migratory capacity of transplanted cells corresponded with their in vitro motility . The current zebrafish embryo injection model has been previously used to assess the metastatic behavior of lung cancer cells [40,64].
In the present study, the in vitro assay of single-cell suspension dissociated from 3D spheroids was performed by the scratching assay. As shown in Fig. S13, SI and Table S5, MIA cells dissociated from 3D spheroids of 1:9 ratio had the farthest displacement (i.e. the straight distance from the farthest point to the start point) in 8 h. These data supported that single-cell suspension dissociated from 3D spheroids remained similar properties as in vivo model. However, the mean ve-locity of the MIA cells showed no significant diﬀerences in the co-cul-ture groups with various ratios. (Fig. S14, SI). Besides, the accelerated movement of MIA cells was observed in MIA-PSC co-spheroids in vitro. According to the fluorescence tracking, the co-cultured MIA cells did
not always move together with PSCs in the zebrafish, suggesting that the migration of MIA cells was increased not only through co-migration with PSCs, but also through the enhanced motility of MIA cells. Fur-thermore, the survival of MIA cells was improved by co-culture with PSCs, in consistence with the literature finding that PSCs oﬀered the suitable microenvironment for cancer cell survival during metastasis . Experimental findings in zebrafish provide additional evidence to support the current MIA-PSC 3D spheroids as an in vitro tumor model for PDAC.