Supplementary Figures 1 - 9 from CAF-Secreted Annexin A1 Induces Prostate Cancer Cells to Gain Stem Cell–like Features
Authors: Geary Lauren A. , Nash Kevin A. , Adisetiyo Helty , Liang Mengmeng , Liao Chun-Peng , Jeong Joseph H. , Zandi Ebrahim , Roy-Burman Pradip 
Published: April 2023

<p>PDF file - 2259KB, Supplementary Figure S1. Phenotypic changes consistent with EMT in cE1 culture with CAF CM. A, The greatest observed amount of EMT-like morphology was seen in SCmed cE1 cells grown with CAF CM treatment after 14 days in 3-D Matrigel assays. Representative bright field images of the observed morphologies are shown. B, CAF CM treatment did not lead to increased ability to form spheroids or undergo morphologic changes akin to EMT in SCnone, compared to SCmed. CAFs 1 and 3 were used in independent repeats. Bar 100 μm. Supplementary Figure S2. Ammonium sulfate precipitation of conditioned media proteins. A, Potential mediators of EMT-CSC lineage present in the CAF CM have been enriched using several rounds of ammonium sulfate precipitation followed by in vitro biological assays for protein activity affecting the EMT16 sensitive fraction of the cE1 cell line, SCmed, using the three-dimensional Matrigel culture system. The 40% AS-enriched fraction from the CAF CM had the most extensive EMT-like morphology after 14 days of culture, compared to vehicle control and NPF control. Bright field images were taken at 40x magnification. Bar 100 μm. B, Biological activity for each CAF CM fraction was assayed using the in vitro spheroid formation assay with cE1 SCmed. In agreement with the data in A, SCmed cells treated with CAF CM 40% AS-fraction displayed the highest propensity toward EMT based on quantitative real time PCR analysis of EMT transcription factors (Snail, Slug and Twist), stem cell transcription factors (Oct4, Sox2 and Nanog) and decrease in E-Cadherin. Ratios are expressed as fold change normalized to β-actin expression. CAFs 1 and 3 were used in independent repeats. Supplementary Figure S3. A, Dose response to AnxA1 nAb in the presence of AnxA1 enriched CAF CM AS fraction. CAFs 1 and 3 were used in independent repeats. B, Fpr-rs1 is found in greatest abundance in cE1 cells. ?, P < 0.05; *, P <0.01; **, P < 0.001. Supplementary Figure S4. Gross morphology of cE1 grafts under renal capsule. Mean graft size was measured by diameter and found to be significantly increased in Ac2-26 treated versus control. *, P < 0.001. Supplementary Figure S5. Effect of AnxA1 mimetic and recombinant forms on spheroid formation of the primary tumor cell fractions in vitro. A, Phase contrast, hematoxylin and eosin (H&E), and immunofluorscence (IF) images show representative LSChi spheroid morphologies under influence of co-culture with UGSM or CAF and treatment with AnxA1 enriched CAF CM AS fraction or Ac2-26. Spheroids from 3 cell cultures of UGSM and CAF co-culture, 3 cultures of AnxA1 enriched CAF CM AS fraction treated, and 5 cell cultures of Ac2-26 treated LSChi were evaluated for this purpose. Co-IF was performed using antibodies against the basal cell marker p63 (red) and luminal cell marker CK8 (green) and DAPI (blue), to label cell nuclei. CAF1 and its fractions were used in at least two independent repeats and, predominantly, CAF3 and its fractions were used for at least one additional independent repeat. No differences between CAFs 1-3 were observed when using these cultures in experiments. Bar 100 μm. Supplementary Figure S6. A, Spheroids were counted for 5 independent cultures of Ac2-26 treated LSChi, 2 independent cultures of Ac2-26 treated LSCmed, and 3 independent cultures of rAnxA1 LSChi and LSCmed (rAnxA1 n=1, 1 and 5 minced prostates). AnxA1 treatment of LSChi and LSCmed cells led to increased numbers of spheroids formed in a dose-dependent manner. Representative bright field images are shown at 40x magnification. Bar 100 μm. B, AnxA1 concentrations increase pErk1/2 activation in LSChi spheroids. Positive control cell lysates, UV-treated NIH/3T3 and T-47D TGF-β1 control lysate (Santa Cruz Biotechnologies), which are indicated as + in western blots, were used. 3.3-fold pErk1/2 activation and 2-fold Erk1/2 activation was detected by western blot analysis in LSChi spheroids after treatment with increasing concentrations of Ac2-26. pErk1/2 was reduced in the presence of Ac2-26 with α-AnxA1 nAb. NS, not significant; *, P < 0.05; ?, P < 0.01; **, P < 0.001. Supplementary Figure S7. Representative immunohistochemistry of control prostate tissue and grafts for Cre and Pten. PP ("wildtype") and CPP (Pten deletion) prostate tissues were stained by immunohistochemistry (IHC) for Cre and Pten expression. PP controls are Cre negative and Pten positive. CPP tumor-burdened prostate tissues are Cre positive and Pten negative. Representative Cre and Pten staining of acinar glandular structures from LSChi + rAnxA1 grafts is shown. Glandular structures seen in grafts are Cre positive and Pten negative. Magnification at 100x and 400x is indicated in figure. Bar 100 μm. Supplementary Figure S8. Histologies of acinar glandular structures in AnxA1 treated LSChi and LSCmed grafts. AnxA1 treated LSChi/med acinar glandular structures are more prolific in regards to size and number than cuboidal structures, have more CK8+ prostate tumor cells, possess p63+ foci and have a lower resulting percentage of p63+:CK8+ cells. Magnification at 400x. Bar 100 μm. Supplementary Figure S9. Analysis of Erk pathway in LSChi/med grafts. A, C, IHC analysis of AnxA1 ligand-receptor pErk1/2 downstream activation in glandular structures. Representative images of pErk1/2 expression in LSChi/med untreated control, Ac2-26 treated and rAnxA1 treated grafts are shown. Five to six glandular structures from tissue sections per group were stained for pErk1/2 expression. Magnification at 400x. Bar 100 μm. B, D, Ratio of pErk1/2+ normalized to total Erk expression) to Ki67+ cells detected in the grafts. Activation of Erk pathway in AnxA1 treated groups was assessed by increased level of Erk1/2 and pErk1/2. LSChi is shown in B, and LSCmed is shown in D. Statistical significance is indicated by *, P < 0.05; ?, P < 0.01.</p>