Fig 2: Cancer and Stromal Cells Respond to Microenvironmental Signals in Bioprinted Tumors(A) H&E staining of pancreatic tumor tissues printed with PSCs and HUVECs in the stromal compartment and HPAFIIs and HUVECs in the cancer compartment. Bioprinted tissues were treated daily with 5 ng/mL TGFß starting 24 h after printing, and all tissues were fixed on day 10. Scale bar represents 500 µm and 100 µm.(B) Average cellular density of the stromal compartment was quantified in ImageJ from 3 203 images each from at least 3 bioprinted tissues in each condition. Error bars represent ± SD. **p < 0.01 by a two tailed t test.(C) Fluorescent images of bioprinted tissues as in (A) for mCherry (green), VIM (red), and CD31 (yellow). Scale bars represent 500 µm.(D) Quantification of the percent of mCherry-positive cancer within the stromal region in untreated and TGFß treated tissues. Shown is the average ± SEM of 3 structures per condition. *p < 0.05 by a two-tailed t test.Full scans were digitally stitched by the appropriate image software as described in STAR Methods.

Fig 3: CD248 expression in the tumor-associated vasculature of UCB and the comparison with clinicopathological parameters. (A) Representative IHC staining to show different CD248 expression levels in HCC, using CD31 as the positive control. Case 1: No CD248 expression (score = 0), Case 2: Positive CD248 expression in =50% of the tumor-associated vasculature (score = 1), and Case 3: Positive CD248 expression in >50% of the tumor-associated vasculature (score = 2). (B) Comparison between CD248 expression and the Ki-67 index. (C) Comparison between CD248 expression and invasive stage. (D) Comparison between CD248 expression and metastasis. (E) Comparison between CD248 expression and clinical stage. Scale bar = 200 µm. Representative images are shown. *P < 0.05; ns, no statistically significant.

Fig 4: Healing effects of isc-Exo and con-Exo on mouse ischemic injury after hind-limb ischemia. (A) Blood flow recovery evaluated by Laser-Doppler perfusion imaging 0, 7, 14, 21 days after surgery. The left side was the ischemia limb and the right side was the normal control limb. Bars indicate 1 cm. (B) The healing effect of blood perfusion recovery was analyzed by the perfusion ratio of the ischemia side divided by that of the normal control side. The isc-Exo group showed better blood perfusion recovery effects compared to the con-Exo group 21 days after surgery. (C) The ischemic limb skin appearance 21 days after surgery. The PBS group showed scarring on the left ischemia limb and the con-Exo group showed swelling in the whole ischemia limb. Meanwhile, the isc-Exo group showed a better recovery. Muscle sections stained by hematoxylin and eosin (D), CD31 immunohistochemistry (E) and CD31 and a-SMA immunofluorescence (F). The arrows indicate the microvessels in the muscle and the bars indicate 100 µm in the pictures. red: a-SMA, green: CD31, blue: DAPI. (G) The capillary density in the ischemia muscles was analyzed by the number of microvessels per cm2. The isc-Exo group showed higher capillary density than the con-Exo group. * indicates P < 0.05.

Fig 5: Immunofluorescence staining of fibroblasts and myofibroblasts in human colonic mucosa.a Left: HHIP+ fibroblasts in normal mucosa labeled by a-SMA (red) and HHIP (green). Arrows point at the cells expressing both markers. Scale bar indicates 100 µm. Right: The correlation between ACTA2 and HHIP expression in each cell. Fibroblasts in the HHIP+ cluster were double positive for both ACTA2 and HHIP. b Double labeling for a-SMA (green) and CD31 (red). Scale bar indicates 100 µm. c Double labeling for a-SMA (red) and Keratin 20 (green). Scale bar indicates 100 µm. d Double labeling for fibroblast markers a-SMA (green) and Decorin (red) in the colonic mucosa. Vertical section (left) and cross section (right) of the colonic mucosa. Scale bar indicates 200 µm (left) and 100 µm (right). e Double labeling for Keratin 20 (green) and Podoplanin (red). Vertical section (left) and cross section (right) of the colonic mucosa. Scale bar indicates 100 µm. f Double labeling for Decorin (green) and CD31(red). Scale bar indicates 100 µm. g Double labeling for Decorin (green) and CDX2(red) in the colonic mucosa. Scale bar indicates 50 µm. h Double labeling for Decorin (red) and Podoplanin (green). Each fluorescent channel was displayed separately. Scale bar indicates 100 µm. DAPI: 4',6-diamidino-2-phenylindole, blue-fluorescent labeling nuclei; a-SMA, a-smooth muscle actin, encoded by ACTA2 gene; Decorin, encoded by DCN gene. Podoplanin encoded by PDPN. HHIP, Hedgehog interacting protein, encoded by HHIP gene. Keratin 20, labeling mature enterocytes. CDX2, Caudal Type Homeobox 2, an intestinal specific transcription factor labeling intestinal epithelial and cancer cell. CD31, Platelet and Endothelial Cell Adhesion Molecule 1, labeling endothelial cells. CD45, Protein Tyrosine Phosphatase Receptor Type C, labeling immune cells.