Implanted biomaterials often fail because they elicit a foreign body response (FBR) and concomitant fibrotic encapsulation. To design clinically relevant interference approaches, it is crucial to first examine the FBR mechanisms. Here, we report the development and validation of infrared-excited nonlinear microscopy to resolve the three-dimensional (3D) organization and fate of 3D-electrospun scaffolds implanted deep into the skin of mice and the following step-wise FBR process. We observed that immigrating myeloid cells (predominantly macrophages of the M1 type) engaged and became immobilized along the scaffold/tissue interface, before forming multinucleated giant cells. Both macrophages and giant cells locally produced vascular endothelial growth factor (VEGF), which initiated and maintained an immature neovessel network, followed by the formation of a dense collagen capsule two- to four-weeks post-implantation. Elimination of the macrophage/giant-cell compartment, by clodronate and/or neutralization of VEGF by VEGF Trap, significantly diminished giant-cell accumulation, neovascularization and fibrosis. Our findings identify macrophages and giant cells as incendiaries of the fibrotic encapsulation of engrafted biomaterials via VEGF release and neovascularization, and therefore as targets for therapy.
Tissue exposure to biomaterials triggers a foreign body response (FBR), a step-wise process consisting of inflammation, wound healing and, if not resolved, end-stage tissue fibrosis and scarring
Clinically, the FBR reaches relevance when instigated by medical devices and/or implants, including sensors, pacemaker, prostheses (for example, hip and knee prostheses, breast implants, heart valves), devices for release of bioactive compounds and, more recently, scaffolds used in tissue engineering and regenerative medicine 3,7 . As detrimental processes, inflammation and fibrosis compromise implant integration and long-term functionality, impairing transplant bioactivity due to the fibrous barrier or biomaterial degradation 2,7 . Therefore, intervention strategies to control the inflammatory phase of the FBR and to limit fibrosis are critical to improve implant integration and long-term function.
Different concepts have been explored to attenuate the FBR and fibrosis experimentally, including improving the biomimetic properties of the biomaterial, for example, by geometrical and/or biochemical surface modifications 19,20 . Likewise, molecular interference has been explored, including: local immunosuppression by corticosteroid deposition 3 ; dampening leukocyte and fibroblast activation by anti-transforming growth factor-antibody or halofunginone 2,21 ; or stimulating vessel development to improve perfusion and performance of the bioactive implants by pro-angiogenic vascular endothelial growth factor (VEGF) 2,3,22,23 . These approaches have resulted in incomplete and often transient efficacy, with ongoing chronic inflammation, fibrosis and even graft failure as outcomes 2,3 . As a potentially stringent approach, the depletion of phagocytes by liposomal clodronate bisphosphonate or genetic ablation reduces the onset of inflammation during an intraperitoneal FBR 19,24 , implicating monocytes/macrophages as early effectors and potential targets of an ill-fated FBR.
Nonlinear imaging of biomaterials
To explore the cause–consequence relationships of the FBR, we developed an intravital window model and applied nonlinear multiphoton microscopy to monitor the fate of engrafted material in mice and in response to molecular intervention. As a currently clinically investigated scaffold design and material platform technology, we applied calcium phosphate-coated medical grade poly(ɛ-caprolactone) (mPCL-CaP) implants, which were fabricated by melt-electrospinning with a fully interconnected honeycomb pore network (average pore size 100 μm, 90% porosity, Fig. 1a)
Scaffold fibres elicited strong second (SHG) and third harmonic generation (THG) signals, using excitation/emission wavelengths of 1,090/545 and 1,180/393 nm, respectively (Fig. 1b). SHG/THG z-scanning resolved both individual fibre geometry in three dimensions (Fig. 1b) and an additional weak THG signal from the calcium phosphate coating (Fig. 1b, arrowheads). Remarkably, SHG-/THG-positive substructures were present throughout the fibre core, with strong individual signal profiles and insignificant co-localization (R 2 = 0.06, P < 0.001, Fig. 1c,d). Thus, SHG/THG signals resolve different structural PCL subdomains after three-dimensional (3D) printing, such as the crystalline phase of the melt electrospun fibres 33 .
To extend the applicability of nonlinear SHG/THG microscopy for other implant types used in clinical applications, we analysed a series of biomaterials next to mPCL-CaP, including polyester, polysulfone (PSU), and biodegradable ones, such as polyglycolic lactic acid (PGLA) and gelatin. All tested materials elicited a strong THG signal, which predominantly originated from the scaffold interface independently from their microarchitecture (Supplementary Fig. 2). Likewise, a SHG signal was emitted by most materials, except PSU (Supplementary Fig. 2). The results establish the THG signal, in particular, as a universal interface-detection strategy for robust analysis across implant types.
As a prerequisite for intravital imaging in animals, we tested whether the SHG/THG signals induced by scaffold fibres were sufficient for reliable detection in the backward direction, which is mandatory for thick tissue microscopy 32 (Fig. 1e,f). Signal intensity was fourfold decreased when detected in the backward, as compared with the forward, direction, and an additional 50% of signal decayed per 25 μm penetration into the fibre for each detection mode (Fig. 1e,f). Despite this scatter-induced signal loss, 40-μm-thick fibres were completely detected in either configuration (Fig. 1e). This establishes the combined SHG/THG signal detection as a tool for the non-destructive analysis of scaffold fibre geometry and of substructures of semicrystalline mPCL polymers.
Intravital longitudinal imaging of the FBR
To monitor both extracellular matrix and cellular kinetics during the FBR in vivo, an mPCL-CaP scaffold of 5 × 5 × 0.2 mm was implanted in parallel to the deep dermis/subcutis interface in GFP-C57BL/6 mice expressing GFP in all cells and in nude (nu/nu) mice, and analysed through an optical imaging window over time (Fig. 2a and Supplementary Fig. 3a). The scaffolds integrated without macroscopic tissue trauma, inflammation or bleeding (Fig. 2b), similarly to the integration of scaffolds in window-free mice 25 . Fourteen days after implantation, a histologically significant FBR response emerged, characterized by a cell-rich infiltrate and multinucleated giant cells decorating the surface of fibres, high density of neovessels and emerging fibrosis between and surrounding the scaffold fibres (Fig. 2c). The composition of the inflammatory infiltrate, which indeed may vary with material type and geometry 19,25,34,35 , was further delineated by flow cytometry; macrophages/giant cells represented the predominant cell type of the immune infiltrate (47.2%), followed by T-cells (20%), granulocytes (9.1%) and B-cells (4%) (Supplementary Fig. 4a). Giant cells and macrophages accumulated with a ratio of about 1:6 and retained monocytic differentiation marker CD68 (Fig. 2d) and M1 polarization (IRF-5, interferon regulatory factor 5), but lacked the M2 profile marker CD163 overtime (up to seven weeks; Fig. 2e and Supplementary Fig. 4b). Thus, a macrophage-dominated pro-inflammatory M1-type response was induced by mPCL-CaP implants during early and late stages of the FBR, with no evidence for M2 polarization over time, in contrast with what occurs with other material types 9,15,16 .
When monitored longitudinally by intravital microscopy, the tissue surrounding the THG-positive mPCL-CaP scaffold became gradually infiltrated by GFP-positive cells (Fig. 2f,g), followed by sprouting of perfused neovessels and deposition of, initially loose and later bundled, fibrillar collagen (SHG) throughout the initially SHG-free wound bed (Fig. 2f,g and Supplementary Fig. 3b,c). The SHG signal intensity in the scaffold fibres was diminished in parallel to progressing collagen deposition, whereas the THG signal intensity remained largely unperturbed (Fig. 2g). This established the THG signal as the most robust detection mode in vivo, to monitor the positioning and integrity of the scaffold over time. Although the FBR kinetics in the GFP-C57BL/6 and nude mice were similar, neovascularization was enhanced in the nu/nu mice, consistent with angiogenic strength being dependent on the genetic background of mouse strains 36,37 . Despite severe inflammatory tissue remodelling, mPCL-CaP fibres were neither resorbed nor infiltrated by cells (Supplementary Fig. 3d,e), confirming the documented stability of this material in vivo 28,38,39.
To demonstrate in vivo applicability of THG imaging for an independent material, PSU implants were efficiently monitored, including early recruitment of GFP-positive cells, robust neovascularization and collagen deposition (Supplementary Fig. 5), similar to the response to mPCL-CaP.
Kinetic and mapping of infiltrating cells
Unlike their composition, the dynamics of FBR infiltrate cells remain unknown. We mapped infiltrate kinetics and the accumulation of foreign body giant cells along the scaffold surface and performed time-lapse recordings and single-cell tracking of infiltrating GFP-positive cells. Relative to the implant position, we identified two kinetic subpopulations that were recruited over time, including interstitially moving and fibre-attached resident cells (Fig. 3a–c and Supplementary Videos 1–3). A population of mononuclear interstitial cells retained mostly kinetic behaviours and moved along and between collagen fibrils, without directly contacting scaffold fibres (Fig. 3a,d; speed up to 3 μm min−1). These were interstitial leukocytes, also detected by histology (Fig. 2c). Conversely, scaffold-attached cells were typically multinucleated, as detected by intravital injection of Hoechst dye (Fig. 3b, arrowhead), with high positional stability despite vigorous cytoplasmic dynamics (Fig. 3d and Supplementary Video 4). These were stably anchored resident giant cells, as predicted 7 ; they nonetheless retained slow dynamic behaviours, locally exploring the material surface by ruffling lamellipodia 4 (Fig. 3b,d; average speed at day 14: 0.07 μm min−1).
Neoangiogenesis and fibrotic capsule development during FBR
Whether neovessels contribute to enhance or resolve the FBR remains unclear 2,3,22,23 . Both immunodeficient and immunocompetent mice carrying mPCL-CaP material developed a dense neovascular network (Fig. 2f,g and Supplementary Fig. 3b,c), with an irregular-shaped morphology, an enhanced number of branch points and a decreased inter-branch length, compared with the linear organization of normal dermal blood vessels (Fig. 4a,b). These features are similar to those of tumour neovessels 40,41 , where dysfunctional angiogenesis supports cancer lesion growth, progression and metabolic deregulation 42 . Neovessels preferentially oriented in parallel or perpendicular to the scaffold fibres, defined by an angle distribution favouring 0 ° and 90 ° (Fig. 4c). This indicated that neovessels sense and undergo guidance by the pre-defined architecture of the scaffold, but thereafter fail to normalize over time, similar to what occurs with dysfunctional vessels in tumour-associated desmoplastic tissue 42 .
In parallel to neoangiogenesis, collagen deposition occurred in an incremental fashion (Fig. 2f,g), histologically forming a fibrillar capsule surrounding and penetrating the scaffold architecture by day 14 and thereafter (Fig. 4d). In a 3D reconstruction, two subsets of SHG-positive collagen fibres and bundles were detected, including fibres wrapping around (60–90 ° orientation) or aligning in parallel (0–15 °) to scaffold fibres (Fig. 4e,f). This indicated fibrous scarring as a mechanically precise process, leading to functionally distinct subsets of collagen bundles. In aggregate, giant-cell development is accompanied by a desmoplasia-like process consisting of leukocyte influx, primordial vascularization, and progressive collagen deposition and encapsulation of the porous implant.
Therapeutic targeting of macrophages and neoangiogenesis
To test whether infiltrating macrophages and multinucleated giant cells are the cause or consequence of neovascularization and late-stage fibrosis, we depleted the macrophage lineage of scaffold-bearing mice with clodronate liposomes before graft implantation. After internalization, clodronate induces apoptosis of phagocytes but no other cells 43 . Clodronate treatment stopped the recruitment of infiltrate cells and giant-cell development, as previously reported 19,24 , and further blocked scaffold-associated neovascularization and fibrosis (Fig. 5a,b and Supplementary Fig. 6a). To directly test whether neovessel development and late-stage scarring were linked and dependent on the presence of macrophages and giant cells, lesions containing the scaffold were stained for VEGF-A, the predominant proangiogenic cytokine in regenerating and cancerous tissue 44 and an important cytokine produced by M1 macrophages 8,9 . VEGF-A was prominently expressed in mononucleated stromal cells and giant cells (Fig. 5c, left panel); after clodronate treatment both VEGF-positive cell subsets were absent, without compensatory up-regulation of VEGF-A in other tissue regions (Fig. 5c, right panel), and there was an absence of neoangiogenesis (Fig. 5a). This identified myeloid and giant cells as the main source for VEGF-A and as drivers of neovessel formation.
We next aimed to clarify whether neovascularization is an independent mechanism that enhances or counteracts the severity of the FBR and scarring. To minimize neovessel establishment directly, without targeting giant cells, we administered VEGF Trap to prevent VEGF availability 45 , using a dosing scheme effective for anti-angiogenic therapy in patients 46,47 . VEGF Trap stopped vessel formation and significantly diminished the numbers of giant cells and collagen deposition (Fig. 5a,b and Supplementary Fig. 6a). VEGF Trap monotherapy was slightly less efficient than clodronate treatment, but combining both agents resulted in near-complete blocking of giant cells and fibrosis development; although, this was not an improvement on the already profound effects observed by clodronate treatment alone (Fig. 5a,b).
When addressed longitudinally by intravital microscopy, clodronate-mediated cell depletion, effective by day 7, preceded the blocking of neovascularization and fibrous encapsulation of scaffold fibres by day 14 (Fig. 5d and Supplementary Fig. 6b). This failure to mount both neovessels and fibrosis after macrophage/giant-cell targeting indicated that myeloid cells are non-redundant drivers of FBR-associated tissue remodelling. VEGF Trap inhibited tissue infiltration by GFP-positive cells after, but not before, day 7 (Fig. 5d), suggesting a two-step process with early-onset inflammation mediated by VEGF-independent pre-existing vessels, followed by secondary exacerbation via VEGF-dependent neovessels.
Cell accumulation near the implant was strongly diminished by clodronate and/or VEGF Trap (Supplementary Fig. 7a,b), but the positional stability of residual scaffold-associated cells and the mobility distribution of interstitial cells remained unperturbed, compared with untreated lesions (Supplementary Fig. 7c–e). These results indicated that both clodronate and VEGF targeting act by limiting cell entry into the tissue rather than mobility within the lesion.
To investigate the long-term efficacy of macrophage and neovessel depletion on the FBR and fibrotic encapsulation beyond the two-week observation period achievable using the window system, window-free mice bearing scaffolds were treated with clodronate liposomes and/or VEGF Trap and assessed by ex vivo 3D SHG/THG microscopy and histology on day 28 (Fig. 6a–c and Supplementary Fig. 6a, lower panels). Suppression of the FBR, including diminished inflammation, vascularization and fibrosis, was sustained by anti-angiogenesis therapy and, with stronger effects, by clodronate and combined clodronate and VEGF Trap treatment (Fig. 6b,c).
Lastly, we tested whether the therapeutic effects persisted after discontinuation of clodronate and/or VEGF Trap administration, by monitoring FBR giant cells, vessels and collagen deposition 14 and 28 days after the treatment was suspended (Fig. 6d). For all therapy settings, the FBR severity remained significantly diminished until at least four weeks post therapy withdrawal, including stable collagen levels and vessel numbers, and a decreased but partly relapsing density of giant cells (Fig. 6e–h). This indicated that dampening the initial inflammatory phase is crucial to reach a sustained anti-angiogenic and anti-fibrotic response.
In summary, by implementing nonlinear intravital microscopy to monitor 3D porous mPCL-CaP scaffolds and the related cell and tissue dynamics, we identified the connection between giant cells and VEGF-induced neovessels as the central pathogenic axis driving the FBR and late-stage fibrosis. In a reciprocal process, implant-associated myeloid cells, including giant cells, locally release VEGF-A, which is required to induce neovessels that, in turn, maintain additional cell recruitment and enhance myeloid inflammation and local encapsulation of the graft site by collagen fibres (Fig. 7). The results suggest that an immature neovessel network aggravates the FBR, similar to the way that tumour-associated neovessels support inflammation, metabolic deregulation and desmoplastic remodelling of the tumour stroma 42 . Because macrophage recruitment, giant-cell development and emerging neovascularization cooperate in series to entertain the fibrosis response, therapeutic interference with either process is suited to limit tissue remodelling and scar formation 24 .
A rich set of cytokines is released during a FBR, including IL-1b, IL-4, IL-6, IL-8, IL-10, IL-13, MCP-1, TGF-β and VEGF 2,7,9,48,49 . However, their pathogenic impact on whether they aggravate or limit late stages (in particular) of the FBR is poorly understood 2 . Besides confirming macrophages and giant cells as central drivers of FBR, we identified a major role for VEGF-releasing M1 polarized cells as important exacerbators of the FBR to mPCL-CaP, with persistent and probably dysfunctional neovascularization and fibrosis as outcomes. Targeting macrophages and giant cells, either by non-specific depletion using clodronate liposome treatment, or by a targeted molecular therapy to remove specific subsets (for example, M1- or M2-skewed cells 50 ), represents a promising strategy to modulate both early inflammation (days), as shown recently 19 , and late-stage tissue remodelling (weeks) to minimize a chronic FBR and detrimental scarring.
Our results further establish anti-VEGF therapy as an intervention principle 48 to efficiently reduce both neovascularization and subsequent macrophage influx. Thus, anti-VEGF therapy might be specified for grafts that do not require sustained vascularization for full functionality, but suffer from inflammation and fibrosis, including implanted prosthesis, valves, pacemakers, catheters and defibrillators 3,7 .
Depleting myeloid cells and neovascularization using targeted (combination) therapy, may produce a largely avascular and non-inflamed integration site, which may be critical towards achieving a scar-free long-term integration. Given their proven safety profile in clinical application, where they are generally tolerated well with no long-term adverse effects on the immune system
Scaffold design and fabrication
mPCL (Purac Biochem) was 3D printed by means of melt electrospinning writing (MEW) 54 . Briefly, mPCL was melted at 94 °C in a glass syringe and extruded through a 23G blunt-end needle at 10 μl h−1 and at 20 mm distance from a rotating metallic collector, which was mounted on an x–y stage under an electrical field of 11–12 kV. Scaffolds were surface-treated with NaOH etching and/or CaP coating to reduce the hydrophobicity of PCL. For NaOH etching, scaffolds were soaked in 70% ethanol for 15 min, washed with distilled water, subjected to a 3M NaOH solution (1 h, 37 °C) and then washed with distilled water until pH 7.0 was reached. CaP coating was performed as described in ref. 54. After surface treatment, scaffolds were sterilized by immersion in 70% ethanol and air dried under exposure to ultraviolet light.
Dorsal skinfold chamber model and mPCL-CaP scaffold implantation
Animal studies were approved by the Institutional Animal Care and Use Committee of The University of Texas, M.D. Anderson Cancer Center and performed according to the institutional guidelines for animal care and handling. Dorsal skin-fold chambers were transplanted onto 10- to 12-week-old C57BL/6-Tg (UBC-GFP) 30Scha/J (Jackson Lab) or athymic nu/nu mice (experimental radiation oncology, MD Anderson), as described 55 . During surgery, a 5 × 5 × 0.2 mm scaffold was surgically implanted into the subcutaneous tissue of either imaging window bearing or window-free mice. This was monitored for up to 14 and 49 days, respectively. For surgery, mice were anaesthetized using Ketamine/Xylazine and the wound was closed by 4.0 polypropylene sutures.
Intravital microscopy, scaffold imaging and image analysis
For intravital microscopy, mice were anaesthetized with isofluorane and stably mounted onto a temperature-controlled platform (37 °C). The FBR elicited by an implanted mPCL-CaP scaffold was monitored using a custom intravital multiphoton microscope (LaVision BioTech) 31 with three Ti:Sapphire lasers (Chameleon-XR, Coherent) and two Optical Parametric Oscillators (APE/Coherent), resulting in a tunable excitation range from 800 to 1,300 nm (Supplementary Fig. 1). Multi-spectral detection was performed using up to five backward or two forward photomultipliers (PMTs) and up to three excitation wavelengths in two consecutive scans, to separate the following excitation and emission channels: GFP (920 nm; 525/50 nm); Hoechst 33342 (920 nm; 450/60 nm), Rhodamine (1090 nm; 595/40 nm), SHG (1090 nm; 525/50 nm), THG (1180 nm; 387/15 nm) and AlexaFluor750 (1180 nm; 810/90 nm).
For intravital detection, long-working distance ×16 NA 0.8 water or ×25 NA 1.05 multi-immersion oil/water objectives (Olympus) were used. Sequential 3D stacks were obtained of 5–10 μm step-size, reaching up to 200 μm penetration depth. Images were acquired in a random fashion within the subcutaneous tissue up to the dermis. Perfused blood vessels were visualized by intravenous injection of Rhodamine- or AlexaFluor750-conjugated dextran (70 kDa; Invitrogen; 1 mg per mouse). mPCL-CaP scaffolds, in vitro, were analysed using SHG and THG imaging.
Digital image processing, segmentation and quantitative analysis
Images were reconstructed, stitched and analysed using FIJI (W. Rasband, NIH). Individual 3D scan fields representing z-projections of 50 to 300 μm were stitched to large-field montages for both overview and detailed analysis. Quantitative analysis of THG, SHG and fluorescent channels was performed on 3D stacks of 360 × 360 μm, with 10 μm step intervals in the z direction. For standardized enumeration of the local density of GFP-positive cells or vascular density, single channel z-stacks were masked, thresholded (default or Li algorithm), converted to binary images and the signal-positive area was obtained. For each sample, the relative fluorescence density was obtained from ten slices per z-stack, averaged and represented as the percentage of the total area. Four to eight independent fields were averaged per implant from at least four mice per condition. The experiment was repeated three times.
For topography-controlled analysis of fibrillar collagen density in association with scaffold fibres, the SHG intensity of 40 × 40 μm regions were quantified as an average from two adjacent slices preceding the appearance of the PCL fibre, as identified by the THG signal (three to five PCL fibres per sample; three mice per group). Each region was masked, thresholded (Li algorithm) and quantified for the signal-positive area fraction. Single cell velocities from time-lapse sequences were obtained by computer-assisted cell tracking (Autozell). For giant-cell counts, any cell displaying ≥2 nuclei was counted as multinucleated. The mean number of nuclei per giant cell was 5.95 ± 2, quantified from 60 random giant-cells; only a minority of giant cells retained only two nuclei.
Mice bearing mPCL-CaP scaffold in the subcutaneous tissue received VEGF Trap (500 ng per week, once per week) starting four days after scaffold implantation. VEGF Trap is a recombinant fusion protein with VEGF-A neutralizing activity combining the binding portions of VEGF receptors 1 and 2, with the Fc region of the human IgG1 46 . Clodronate liposomes (200 μl per mouse) were administered every two to three days, starting three days before scaffold implantation to deplete macrophages by the day of implantation. For combination therapy, both regimens were combined. For intravital microscopy, three independent fields were averaged per implant, with three mice per group. The experiment was repeated twice.
Mice were euthanized 14, 28, 35 or 49 days after implantation of the scaffold. Scaffold-bearing skin was excised, fixed (4% buffered formaldehyde) and embedded in paraffin for hematoxylin and eosin (H & E) or Masson’s Trichrome staining (five sections per sample, 5 μm thick, three to four samples per treatment). The experiment was repeated twice.
Infiltrate cells associated with the FBR in vivo were isolated and phenotyped by flow cytometry (LSRII FACS, Becton-Dickinson; Diva). To reach sufficient cell numbers, 20 scaffolds were recovered 14 days post implantation, mechanically disaggregated to generate a single-cell suspension and pooled. Phenotyping was performed using the following rat monoclonal antibodies (BD Pharmingen): PE-Cy 7 anti-CD45; PerCP-Cy 5.5 anti-CD11b; Pacific Blue anti-Ly6G, APC-conjugated anti-F4/80 and anti-CD8b. Subset gating and analysis were performed using FLowJo10.2.
All the relevant data supporting the findings of this study are available within the paper, its Supplementary Information and from the corresponding authors upon reasonable request.
How to cite this article: Dondossola, E. et al. Examination of the foreign body response to biomaterials by nonlinear intravital microscopy. Nat. Biomed. Eng. 1, 0007 (2016).
We thank M. Starbuck, C. Johnston, Y. Xiaoqing, R. Jimenez and J. Douglas for histological processing of the samples; and E. De Juan Pardo for the manufacturing of the mPCL-CaP scaffolds. E.D. was supported by the Cancer Prevention and Research Institute of Texas (RP140482) and the Prostate Cancer Foundation (16YOUN24). P.F. was supported by the Netherlands Science Organization (NWO-VICI 918.11.626), the European Research Council (ERC-CoG DEEPINSIGHT, Project No. 617430) and the Cancer Genomics Center, The Netherlands. This work was further supported by the National Health and Medical Research Council (NHMRC Project Grant 1082313), the National Breast Cancer Foundation (NBCF IN-15-047) and the Worldwide Cancer Research (WWCR 15-11563) to B.M.H. and D.W.H. and the German Research Foundation (DFG HO 5068/1-1) to B.M.H. The Genitourinary Cancers Program of the CCSG shared resources at the MD Anderson Cancer Center was supported by the National Institute of Health/National Cancer Institute award number P30 CA016672.