br collagen gel contraction Using in vitro
collagen gel contraction. Using in vitro Transwell Matrigel invasion assay, we observed increased invasion of A549 tumor Bafilomycin A1 into Matrigel in co-culture with HD-CAFs but not with LD-CAFs or A549 tumor cells alone (Figure 3B and Supplementary Table S9). Furthermore, in vitro co-culture migration assay showed increased migration of A549 tumor cells in co-culture with HD-CAFs but not with LD-CAFs or alone (Figure 3B and Supplementary Table S9). To confirm aggressiveness of high desmoplastic CAFs, we did an in vivo tumor growth assay. In addition to selected CAFs used in in vitro invasion and migration assay, we included two HD-CAF and two LD-CAF primary cultured cells and their matching NFs based on collagen remodeling. Both HD- and LD-CAF cells were separately co-injected subcutaneously with A549 tumor cells into SCID mice. The tumor growth rate for all in vivo tumor-promoting curves of eight pairs of CAFs and NFs co-injected with A549 tumor cells including A549 tumor cell alone as control was analyzed using the mixed-effect model. The tumor growth rate in the HD-CAFs/A549 was significantly higher than in LD-CAFs/A549 and NFs/A549 (P b .0001; Supplementary Table S10), but there was no statistical difference between LD-CAFs/A549 versus NF/A549 (P = .66; Figure 3C). We did not observe any tumor growth when we injected HD-CAFs, LD-CAFs, or NFs alone. We observed the slowest tumor growth rate in tumor from injecting A549 tumor cells alone. This implies that the increased tumor growth rate observed in HD-CAFs/
Figure 2. The association between desmoplasia and the RR was tested for 165 patients. (A and B) The criteria for desmoplasia as described in Figure 1 were used to assess desmoplasia in 165 tumors from UHN NSCLC patients. (C) The RR was estimated using the cumulative incidence function, and the comparison between 165 HD and LD tumors was performed using Wald test within the Fine and Gray model. (D) The association between desmoplasia and the RR within adenocarcinoma subgroup was tested for 116 patients. The RR was estimated using the cumulative incidence function, and the comparison between HD and LD tumors was performed using Wald test within the Fine and Gray model. r> A549, LD-CAFs/A549, and NFs/A549 was due to the promoting ability of fibroblasts. These data indicated that high level of desmoplasia is associated with tumor aggressiveness, independent of the CAF number, thus suggesting that the aggressiveness of the tumor depends more on CAF function rather than CAF density.
HD EX-CAFs Enhance Invasion of NSCLC Tumor Cells We then aimed to understand the molecular mechanisms of high desmoplastic CAFs in tumor aggressiveness. We focused on collagen matrix remodeling where differences in gel contraction between HD-and LD-CAFs indicated that Lumen are behaving in a functionally different manner. Therefore, HD-CAFs and LD-CAFs were clustered based on their correlation between collagen matrix remodeling activity (collagen gel contraction) and desmoplasia (Supplementary
Table 1. The Effect of Desmoplasia on Clinical Outcome
RR was adjusted for clinical factors in the UHN cohort and adenocarcinoma patient subgroup when the model was weighted for the amount of stroma. The Fine and Gray model was utilized. Ade, adenocarcinoma.
Figure S4). We noticed an overlap of HD-CAFs and LD-CAFs when the DA score ranged from 20% to 60%. Therefore, we selected three extreme HD-CAFs (HDEX-CAFs; desmoplasia N60% and collagen gel diameter b median) and four extreme LD-CAFs (LDEX-CAFs; desmoplasia b20% and collagen gel diameter N median) to better define CAF subgroups for functional analysis. In vitro 3D collagen matrix invasion assay showed increased invasion of A549dsRed tumor