Patient-derived Models Reveal Impact of the Tumor Microenvironment on Therapeutic Response  Ayesha A. Shafi, Matthew J. Schiewer, Renée de Leeuw, Emanuela Dylgjeri, Peter A. McCue, Neelima Shah, Leonard G. Gomella, Costas D. Lallas, Edouard J. Trabulsi, Margaret M. Centenera, Theresa E. Hickey, Lisa M. Butler, Ganesh V. Raj, Wayne D. Tilley, Edna Cukierman, Karen E. Knudsen  European Urology Oncology  Volume 1, Issue 4, Pages 325-337 (September 2018) DOI: 10.1016/j.euo.2018.04.019 Copyright © 2018 European Association of Urology Terms and Conditions

Fig. 1 The patient-derived explant (PDE) model of prostate cancer sustains tumor morphology, viability, and endogenous endocrine signaling. (A) Top: Method used to culture patient tumors ex vivo. Drug treatment can be added to the medium to investigate the effect on tumor growth. Media (and appropriate treatments) were replaced every other day. Tissue was harvested and fixed in 4% formalin. The formalin-fixed tissue was then embedded into paraffin blocks and cut into sections with a microtome. Bottom: Slides were stained with hematoxylin and eosin (H&E). The representative images shown (10× and 40×) indicate maintenance of gross tumor morphology after culturing ex vivo for up to 6 d. (B) PDE samples stained for HIF1α showed that the tumors received sufficient oxygen supply to maintain viability. (C) Androgen receptor (AR) and prostate-specific antigen (PSA) immunostaining of tissue (patient #5) demonstrated sustained endogenous and endocrine signaling in the explants after 6 d of ex vivo culture. All immunohistochemistry images are shown at magnification of 40×. (D) PSA secreted into PDE media was analyzed on days 2, 4, and 6 in hormone-proficient media (squares) and hormone-deficient media (diamonds). (E) Expression of AR and AR target genes (TMPRSS2, PSA, and FKBP5) in four different PDEs, shown as relative expression normalized to 18S. Tumor tissue was matched to the corresponding non-neoplastic tissue control. European Urology Oncology 2018 1, 325-337DOI: (10.1016/j.euo.2018.04.019) Copyright © 2018 European Association of Urology Terms and Conditions

Fig. 2 Tumor cells in patient-derived explants (PDEs) exhibit de novo proliferative capacity. (A) Tissue was cultured in complete medium and harvested every 48h for up to 6 d. Ki67 staining was performed to quantify proliferation in the explants. Images are shown at 10× magnification. (B) Ki67 immunostaining revealed a time-dependent increase in proliferation. PDE-3 is shown in panel A. * p<0.05. (C) 5-Bromo-2-deoxyuridine (BrdU) uptake is similar to Ki67 staining. Images are shown at 20× magnification (n=4). European Urology Oncology 2018 1, 325-337DOI: (10.1016/j.euo.2018.04.019) Copyright © 2018 European Association of Urology Terms and Conditions

Fig. 3 Androgen receptor (AR) signaling can be perturbed both genetically and pharmacologically in the patient-derived explant (PDE) model. (A) Knockdown of AR was achieved via lentirviral transduction of PDE with an AR-directed shRNA for 6 d. Representative images show AR immunohistochemistry for shCon and shAR after 6 d. Right: mRNA expression of AR and AR target genes (PSA, FKBP5, and TMPRSS2) from tumor samples treated with shCon and shAR for 2, 4, and 6 d (n=3), with fold change for shAR versus shCon. Con=control. (B) Representative images (10× and 40× magnification) for tumors treated with vehicle, an AR antagonist (1μM enzalutamide), and an AR antagonist (1μM bicalutamide) for 6 d (n=3). (C) mRNA expression of AR target genes (PSA, TMPRSS2, and FKBP5) in PDE treated with vehicle, 1μM enzalutamide, and 1μM bicalutamide. The average fold change for three PDEs is shown. * p<0.05. (D) Waterfall plot depicting the percentage change in PSA in the culture medium with 1μM enzalutamide treatment. Blue bars represent patients with a ≥25% decrease in PSA, black bars represent no change in PSA, and red bars represent a ≥25% increase in PSA. European Urology Oncology 2018 1, 325-337DOI: (10.1016/j.euo.2018.04.019) Copyright © 2018 European Association of Urology Terms and Conditions

Fig. 4 The patient-derived explant (PDE) model is responsive to clinically approved and experimental prostate cancer therapeutics. Tissue was cultured in complete medium, treated with different drugs, and harvested after 6 d. Drug treatment was changed out every 48h. Ki67 staining was performed to determine the amount of proliferation that occurred to evaluate the effect of drug treatment on PDE growth. (A) Representative hematoxylin and eosin (H&E; 10× magnification) and Ki67 (40× magnification) images are shown for PDE-14 tumor samples treated with vehicle, an androgen receptor (AR) antagonist (1μM enzalutamide), taxane (50nM docetaxel), and an AR antagonist (enzalutamide) + taxane (docetaxel). (B) Quantification of Ki67 immunostaining in three separate patient samples. PDE-14 is the sample used for the images in A. (C) Representative H&E (10× magnification) and Ki67 (40× magnification) images for samples treated with vehicle, a PARP inhibitor (2.5μM veliparib; PDE-17), a CDK4/6 inhibitor (1μM palbociclib; PDE-21), and a DNAPK inhibitor (1μM NU7441; PDE-22). (D) Quantification of Ki67 immunostaining for three separate patient samples, including the samples used for images in C. European Urology Oncology 2018 1, 325-337DOI: (10.1016/j.euo.2018.04.019) Copyright © 2018 European Association of Urology Terms and Conditions

Fig. 5 Initial tumor-permissive stromal pattern in the patient-derived explant (PDE) model indicates possible resistance to enzalutamide. (A) Schematic explaining tumor microenvironment (TME) characteristics indicative of a permissive stromal type and representative simultaneous multiplex immunofluorescence image of masks (cytokeratin [Cyto], vimentin [Vim], and nucleus [Nuc]) in non-neoplastic tissue on day 0 (N), tumor on day 0 (T), and tumor tissue treated with either vehicle (CTL) or enzalutamide (ENZ) for 6 d. The tumor example has a tumor-permissive TME with desmoplastic traits, displaying high pFAK and high active α-5-integrin. (B) Quantification of the markers assessed in stromal areas: pFAK and active α5-integrin. (C) An additional sample with a tumor-permissive stromal pattern. (D) Ki67 staining was performed to quantify proliferation to evaluate the effect of drug treatment on PDE growth. Representative Ki67 images (40× magnification) and quantification are shown for PDE samples treated with vehicle and the AR antagonist enzalutamide (n=2). European Urology Oncology 2018 1, 325-337DOI: (10.1016/j.euo.2018.04.019) Copyright © 2018 European Association of Urology Terms and Conditions

Fig. 6 Enzalutamide reinforces the tumor-restrictive desmoplastic stromal pattern in the patient-derived explant (PDE) model of prostate cancer. (A) Schematic explaining the tumor microenvironment (TME) characteristics indicative of tumor-restrictive stromal type and representative immunofluorescence images of masks (cytokeratin [Cyto], vimentin [Vim], and nucleus [Nuc]) in non-neoplastic tissue on day 0 (N), tumor on day 0 (T), and tumor tissue treated with either vehicle (CTL) or enzalutamide (ENZ) for 6 d. The tumor example has a tumor-restrictive TME with desmoplastic traits, displaying low pFAK. (B) Quantification of the markers assessed in stromal areas: pFAK and α5-integrin. (C) Additional samples with a tumor-restrictive stromal pattern. (D) Ki67 staining was performed to quantify proliferation to evaluate the effect of drug treatment on PDE growth. Representative Ki67 images (40× magnification) and quantification are shown for PDE samples treated with vehicle and the AR antagonist enzalutamide (n=3). European Urology Oncology 2018 1, 325-337DOI: (10.1016/j.euo.2018.04.019) Copyright © 2018 European Association of Urology Terms and Conditions