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wrap them (Videos S4, S5, S6, and S7, SI). Both MIA Cycloheximide and PSCs in co-culture actively migrated on CS-HA coated plates, especially when the ratio of PSCs increased (Fig. S6, SI). The moving patterns (trajec-tories) of MIA cells alone and PSCs alone are shown in Fig. 3B. The moving patterns of co-spheroids in diﬀerent seeding ratios are shown in Fig. 3C and co-spheroids formed more quickly as shown in Fig. 3D. Based on the cell trajectories, the 1:9 co-cultured MIA-PSCs underwent collective self-assembly, where the MIA cells migrated collectively to become the core and were surrounded by PSCs to form co-spheroids. Table S3 provides the averaged values for the cell moving speed in 8 h (mean velocity in μm/min). The migration rate of cancer cells in spheroids (from 1:9 cell ratio) was ∼1.167 μm/min, which was much faster (3.2-fold) than that of cancer cells alone (∼0.361 μm/min) on the same coated plates. Data revealed that Supplementary Fig. S6. When the initial population of PSCs was higher, MIA cells in the 1:9 co-cul-tured group moved more rapidly than the other groups.
3.3. Gene expression for cells cultured on TCPS (2D) or CS-HA coated plates (3D)
The expression levels of several tumor-associated genes were ana-lyzed to determine the tumorigenicity of tumor-like co-spheroids (Fig. 4A). The typical markers for epithelial to mesenchymal transition (EMT), including E-cadherin (E-cad), N-cadherin (N-cad), Vimentin, TWIST1, and SNAIL, were analyzed at 48 h. For mono-cultured MIA cells, the gene expression levels of E-cad, N-cad, TWIST1, and SNAIL revealed no significant diﬀerence on CS-HA (3D) or TCPS (2D). The gene expression of TWIST and SNAIL of mono-cultured PSCs was in-creased in about 5.9- and 4.1-fold, respectively, on CS-HA plates vs. TCPS control. The E-cad expression of co-cultured MIA-PSCs were sig-nificantly down-regulated for more than 8.5-fold, while N-cad and Vi-mentin expressions were significantly up-regulated for more than 1.5-and 1.1-fold on CS-HA plates (vs. TCPS). The increase in the expression level of matrix metalloproteinase-2 (MMP2) in co-cultured MIA:PSC (1:9) on CS-HA was increased about 2.4-fold (vs. TCPS). To understand the regulation of ECM genes in MIA-PSC spheroids, the gene expressions of collagen 1 (COL1), LUMICAN, and Sushi, Nidogen and EGF like domains 1 (SNED1) were analyzed. As shown in Fig. 4B, the expression levels of COL1, LUMICAN, and SNED1 for MIA-PSC spheroids on CS-HA (3D) were significantly increased vs. TCPS control by 1.1-, 4.2-, and 4.8-fold, respectively. Meanwhile, COL1, LUMICAN, and SNED1 were up-regu-lated for mono-culture PSCs on CS-HA by 1.6-, 3-, and 3.5-fold vs. TCPS control. The stemness phenotype of spheroids was further investigated. As shown in Fig. 4C, after 48 h of culture, the expression levels of stemness markers CD44 and OCT4 were decreased by ∼1.2- and ∼2.5-fold for mono-cultured MIA cells on CS-HA vs. TCPS control. Mean-while, the gene expression levels of OCT4 for MIA:PSC (1:9) co-spher-oids on CS-HA were increased to 2.5-fold vs. TCPS control. In contrast, CD44 was down-regulated by 1.4-fold for MIA:PSC (1:9) co-spheroids on CS-HA vs. TCPS control. The ATP Binding Cassette Subfamily C Member 1 (ABCC1) and dopamine and CAMP-regulated neuronal phos-phoprotein 32 (DARPP-32) as chemo-resistance related genes were analyzed for their contributions to the increased cell survival in MIA-PSC spheroids against chemotherapy. The gene expression levels of ABCC1 and DARPP-32 for cells on CS-HA were higher than those on TCPS (Fig. 4D). Especially, the expression level of ABCC1 for MIA:PSC
(1:9) co-spheroids on CS-HA was increased to 2.5-fold vs. TCPS control. The expression level of DARPP-32 for PSC homo-spheroids on CS-HA was increased to 12-fold vs. TCPS control.
3.4. Drug eﬀect in tumor-like spheroids
To test whether the microenvironment provided by CS-HA coated plates promoted drug resistance of co-cultured MIA cells and PSCs to chemotherapies, the chemosensitivity assay to gemcitabine was per-formed (Fig. S7, SI). After the treatment of gemcitabine (100 nM) on TCPS for 48 h, the cells were rounded and detached from the plates (Fig. S7A, SI). The IC50 values for MIA cells and PSCs cultured on TCPS (2D) were ∼107 nM and ∼38 nM, respectively (Fig. S7B, SI). On CS-HA coated plates, the IC50 value for MIA cells was about ∼100 nM. Moreover, spheroids from mono-cultured PSCs and from co-cultured MIA:PSC (1:9) on CS-HA plates remained 79.9% and 55.8% at the gemcitabine concentration of 20 μM (Fig. S7C, SI). The PSC homo-spheroids and the co-cultured spheroids also maintained their integrity after gemcitabine (100 nM) treatment. Co-cultured (1:9) spheroids treated with the combination of gemcitabine (100 nM) and Abraxane (12.5 ng/ml) lost their integrity, became smaller, and started to dis-sociate (Fig. 5A). Meanwhile, PSC homo-spheroids remained round and compact even when treated with the combination of gemcitabine (100 nM) and a higher concentration of Abraxane (62.5 ng/ml). In contrast, 2D co-cultured MIA cells and PSCs on TCPS were sensitive to the treatment of Abraxane or a combination of gemcitabine and Abraxane (Fig. 5B and Fig. S8A, SI). Quantitative analyses showed that cells in PSC homo-spheroids had ∼76% viability, and were highly re-sistant to Abraxane or the combination treatment (Fig. 5C). Cells in MIA:PSC (1:9) co-spheroids were sensitive to Abraxane or a combina-tion of gemcitabine and Abraxane, with cell viability down to 29% at 500 ng/ml Abraxane or 40% upon the combination treatment with 100 nM gemcitabine (Fig. 5C and Fig. S8B, SI). Taken together, the MIA-PSC spheroids obtained on CS-HA coated plates revealed sensi-tivity to the treatment of Abraxane or combined gemcitabine/ Abraxane, which is the current clinical drug for pancreatic cancer pa-tients.