Fig 2: Kynurenine and IGF-I pathways were activated in CEFs. (A) Simplified schematic of the kynurenine pathway. (B) Cell lysates of NF-54, CAF-54, and CEF-54 were subjected for immunoblot with indicated antibodies. (C) Western blots in NF-54 cell lysates after treatment with CAF-CM for indicated period. Right: The bands indicate the protein(s) incorporated KYN. (D) The amount of KYN in each CM was examined by ELISA. *P < 0.05 (n = 4. t-test). The data in bar charts are presented as the mean ± SD. (E) NF-54 cells were incubated with recombinant ASPN (20 ng·mL-1). Cell lysates were prepared and subjected to western blot. The band intensities were quantified and normalized to a-tubulin expression, then, expressed as the relative ratio to the control untreated cells in each panel. (F) CD8+ T cells were incubated with NF-54-CM or CEF-54-CM for 2 days and subjected to FACS analysis. Dot plots of 7-AAD and CD8 (left), and histogram of 7-AAD (right). In dot plots, the numbers indicate the percentage of the cells in each fraction. (G) Biotin-labeled NF-54 cells were cocultured with CAF-54 cells for 3 days. Biotin-labeled NFs were isolated by streptavidin-coupled microbeads and cocultured with CD8+ T cells for 2 days. CD8+ T cells were collected, and dead cells (arrow) were detected by 7-AAD staining. (H–M) ASPN was stably overexpressed in NF-50 (H, I) or HSC-43 cells (J–M). (H, J) Western blot analysis of each molecules in cell lysates of parent (pt) cells or ASPN-expressing cells. The value below the KMO panel indicates the ratio of the band intensity to the control after normalized to a-tubulin expression. (K) The amount of KYN in each CM was examined by ELISA. **P < 0.01 (n = 4. t-test). The data in bar charts are presented as the mean ± SD. (I, L, M) CD8+ T cells were incubated with each CM for 2 days and subjected for FACS analysis without fixation. Representative results of three independent assays are shown. Dead cells were evaluated by PI, 7-AAD, or Annexin-V staining, and indicated by arrows in the histogram. (M) +NLG-919 (pre); CM was prepared from HSC-43 ASPN cells pretreated with an IDO-1 inhibitor (5 µm). As a control, NLG-919 was directly added to CD8+ T cells together with HSC-43 ASPN-CM (+NLG-919, post). Boxed area indicates dead CD8+ T cells. (N) PAPP-A-mediated liberation of IGF-I. (O) Western blot of COS-1 cells transfected with IGFBP-4-HA. (P) In vitro cleavage of IGFBP-4-HA by cell lysates of NFs or CEFs was examined. Cleaved and uncleaved IGFBP-4-HA (arrow and arrowhead, respectively) were detected by immunoblot with anti-HA antibody. Longer exposure of cleaved IGFBP-4-HA is shown at the bottom. (Q) Activation of IGF-IR signaling in cancer cells by IGF-I/IGFBP-4 plus CEF-54-CM was examined as described in Section 2.8. (R) Immunoblot of PAPP-A in CEFs and cancer cells. (S) Activation of IGF-IR in OCUM-12 or Li-7 cells was detected by anti-phospho-IGF-IRß antibody. CEF(-) indicates that the IGF-I/IGFBP-4 mixture was directly added to cancer cells. Right panels (+CEF-CM): Activation of IGF-IR was not induced by CEF-CM alone.

Fig 3: (A) The expression and localization of the potential cytokines secreted by huMSCs in the HA-GEL transplantation group and the huMSCs/HA-GEL transplantation group; inserted overview pictures show a lower magnification. (B) The optical density (OD) value of IGF-1, EGF, BDNF, VEGF, and HGF 2 months after HA-GEL transplantation or huMSCs/HA-GEL co-transplantation. (C1) Comparison of IFN-? mRNA expression between the HA-GEL transplantation group and the huMSC/HA-GEL transplantation group. (C2) Comparison of IL-4 mRNA expression between the HA-GEL transplantation group and the huMSC/HA-GEL transplantation group. (C3) Comparison of IGF-1 mRNA expression between the HA-GEL transplantation group and the huMSC/HA-GEL transplantation group. (C4) Comparison of EGF mRNA expression between the HA-GEL transplantation group and the huMSC/HA-GEL transplantation group. *P < 0.05, **P < 0.01, and ***P < 0.001 versus 2 months post–HA-GEL; the results shown are the mean ± SEM of three technical replicates from each animal.

Fig 4: Model of sustained expansion of pro‐tumor fibroblasts by CEFs. (A) CAFs induce ROS production in NFs, which activates the NF‐κB pathway. NF‐κB upregulates HIF‐1α and COX2, which mutually activate one another in a positive feedback loop. ASPN is transcriptionally upregulated by NF‐κB and promotes CD44/Rac1‐mediated cancer cell dissemination, suppresses CD8+ T cell through kynurenine pathway, and activates IGF‐I signaling via PAPP‐A. CEF‐secreted IL‐1β also activates Rac1‐dependent cancer cell dissemination. (B) Model of CEF‐mediated cancer cell dissemination. CAF is generated through the education of resident NFs or bone marrow mesenchymal stem cells by cancer cells. CEFs (blue) sequentially educate resident NFs to further amplify pro‐tumor fibroblasts in the TME, which expands tumor and triggers cancer cell dissemination.