Fig 2: Bioprinted Tissues Can Model Distinct Tumor Cell Subtypes(A) Representative H&E images of tissues bioprinted with luminal (MCF-7), HER2AMP (SKBR3), basal-like (HCC1143), or claudin-low (MDA-MB-231) breast cancer cell lines. All tissues were printed into a stromal mix of HMFs and HUVECs and fixed on day 10. Scale bars represent 500 µm. Representative of 3 independent experiments.(B) Trichrome staining of tissues as described in (A). Scale bars represent 500 µm.(C) Immunofluorescence on sections from bioprinted tissues, as described in (A), stained for KRT8/18 (green), VIM (red), and CD31 (yellow). Scale bars represent500 µm.(D) Luciferase quantitation of relative cancer cell number in bioprinted tissues, as described in (A), over time. Shown is the average ± SEM of at least 5 structures for each cell type representing two independent experiments. MDA-MB-231 endpoint luminescence is significantly different than the other 3 cell lines. ***p < 0.001 from one-way ANOVA.(E) Quantitation of Ki67-positive cancer and stromal cells as assessed from immunofluorescence (IF) images. Shown is average ± SEM of at least 3 tissues at each time point, except for HCC1143 at day 7 (2 tissues). Representative images are shown in Figure S2A. ***p < 0.001; **p < 0.01; and *p < 0.05 by two-way ANOVA.(F) Quantification of Ki67-positive cells in MCF-7, SKBR3, HCC1143, or MDA-MB-231 cells grown in standard 2D culture. Tukey boxplot of two individual experiments with 6 technical replicates per experiment.(G) Quantitation of the cancer cell closeness centrality score in at least 7 tissues for each cell line over two independent experiments. Shown is the average ± SEM. Representative images and masking is shown in Figure S3A. *p < 0.05 by a one-way ANOVA.(H) Quantitation of trichrome staining in bioprinted tissues from (A). Shown is average ± SEM of at least 6 tissues over two independent experiments. *p < 0.05 and **p < 0.01 by a two-tailed t test.Full scans were digitally stitched by the appropriate image software as described in STAR Methods. See also Figures S1, S2, and S3.

Fig 3: Bioprinted Tissues Generated from Primary Patient Tumors Recapitulate In Vivo Morphology(A–C) IF for KRT8/18 (green) and VIM (red) with DAPI (blue) nuclear stain on bioprinted tissue with PSCs and HUVECs in the stromal compartment and disassociated primary patient tumor tissue from OPTR4086 in the cancer compartment (OPTR4086-Bioprint) (A), primary patient tissue from OPTR4086 (B), and PDX tumor tissue generated from OPTR4086 (C). Arrow indicates luminal space. Scale bars for panels (A)–(F) represent 50 µm.(D) IF for E-cadherin (E-cad) (green) and Ki67 (red) with DAPI (blue) nuclear stain.(E and F) IF for KRT8/18 (green) and VIM (red) (E) or KRT8/18 (green) and pS6 (red) (F) with DAPI (blue) nuclear stain in OPTR3099-PDX-Bioprint tissue (PSCs and HUVECs in the stromal compartment with disassociated PDX tumor tissue generated from OPTR3099 in the cancer compartment), primary patient tissue from OPTR3099, and PDX tumor tissue generated from OPTR3099 (OPTR3099-PDX).(G) IF for KRT8/18 (green), VIM (red), and Ki67 (blue) with DAPI (gray) nuclear stain from tissues printed as in (E). Quantification of the percentage of Ki67+/KRT8/18+ dual positive cells shown on right. Average of 3 independent structures shown for OPTR3099-PDX-Bioprint and 3 randomly selected 10× fields for OPTR3099 and OPTR3099-PDX tumor tissue.(H–J) Bioprinted tissue generated as in (E) and treated for 3 days with vehicle (H) or 50 µM (I) or 100 µM (J) of gemcitabine starting at day 4 in culture. IF for KRT8/18 (green) and VIM (red) with DAPI (blue) nuclear stain on bioprinted tissues at day 7 (3 days of treatment). Scale bars represent 500 µm. White box denotes insets of panels (H)–(J) shown below. n = 2–3 structures per condition. Scale bars represent 200 µm.Full scans were digitally stitched by the appropriate image software as described in STAR Methods. See also Figure S7.

Fig 4: 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 5: Distinct Microenvironments Can Be Modeled in Bioprinted Tissues(A) Representative H&E staining of day 10 MCF-7 breast cancer tissues without (top panel) or with (bottom panel) MSCs in the stromal compartment at a ratio of50% HMFs, 25% HUVECs, and 25% MSCs.(B) Representative IF images of day 10 MCF-7 breast cancer tissues as in panel A stained for KRT8/18 (green), VIM (red), and CD31 (yellow).(C) Average luciferase readings over time in MCF-7 tissues printed as in (A) ± SEM. *p < 0.05 and **p < 0.01 by a two-tailed t test.(D) Quantitation of the percentage of Ki67-positive cells in MCF-7 tissues printed as in (A). Shown is the average ± SEM of at least 8 tissues representing two independent experiments that were analyzed at each time point.(E) Representative Masson’s trichrome staining of bioprinted tissues as in (A), fixed on day 4, 7, or 10. Scale bars represent 100 µm.(F) ImageJ quantitation of trichrome staining on tissues from (E). Shown is the average ± SEM of at least six bioprinted tissues per time point in each condition across two independent experiments. *p < 0.05 and **p < 0.01 by a two-tailed t test.(G) qRT-PCR for collagen genes in tissues printed as in (A) (n = 3 tissues), relative to the average “no MSC” value. *p < 0.05 by a two-tailed t test. ns denotes not significant.(H) Digitally stitched second harmonic generation (SHG) images of day 7 bioprinted tissues in a separate MCF-7 print with and without MSCs. Purple color represents signal of mature collagen. n = two tissues per condition.Full scans were digitally stitched by the appropriate image software as described in STAR Methods. See also Figure S4.