Supplement Materials and methods

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1 Supplement Materials and methods Study design and justification of inclusion criteria: We conducted a phase 1/2 pilot clinical trial of intracavernous injections of autologous bone marrow mononucleated cells (BM-MNCs) to treat postradical prostatectomy erectile dysfunction (prp-ed) (NCT ). The study was approved by the appropriate ethics committee (approval number CPP ), and registered on clinicaltrials.gov (#NCT ). The study protocol complied with the Declaration of Helsinki. Written informed consent was obtained from all patients before study enrolment. The study was conducted between 2010 and 2012 at the Henri Mondor Hospital of Créteil. We included 12 consecutive patients with prp-ed, in the first stage of a two-stage design study. The results of this first stage will determine the number of patients required in the second stage of the study. In this first stage, to ensure a tolerance of at least 60%, with a power of 90%, three patients were needed at each escalating dose of BM-MNCs (n = Logβ/Log[1-π], where π is the level of tolerance expected). No serious adverse effects were accepted. Expected serious adverse events were prolonged priapism requiring surgical treatment, pulmonary embolism, or any cardiovascular event. Each patient received a single BM-MNC injection into the cavernous body. Each of four escalating BM-MNC doses was tested in three patients, in numerical order: 2x10 7, 2x10 8, 1x10 9, and 2x10 9 BM-MNCs. After completion of the treatment in each dose group, any adverse events were reported to a Data and Safety Monitoring Board. The studystopping rule was either one serious adverse event or three nonserious adverse events related to the BM-MNC injection. We used a treatment that had never been administered to human patients; therefore, in the present study, it was necessary to use a small number of patients before envisaging a controlled 1

2 phase 2 study. In order to detect the beneficial effects of the BM-MNC injections, we selected patients in whom the spontaneous recovery of erectile function was unlikely. Thus, we included patients with resistance to maximal medical treatment and vasculogenic prp-ed (ie, venoocclusive dysfunction and/or arterial insufficiency) as described below. We considered that these inclusion criteria indicated a very low likelihood of recovering sexual function with or without erectogenic pharmacotherapy, regardless of the status of penile neurovascular bundle preservation indicated in the operating report of radical prostatectomy (RP). Attempts to preserve penile neurovascular bundles during RP does not rule out the risk of irreversible erectile dysfunction, as it is clearly established that even in the hands of experience surgeons, severe damage to cavernous nerves and arteries may occur inadvertently [1 3]. It is therefore not possible to rule out severe penile neurovascular damages solely on the operating report of RP. Instead, we considered that the assessment of penile vascular status by color duplex Doppler ultrasound (CDDU) and the response to erectogenic drugs were robust and pertinent criteria to estimate the odds of recovery of erections. Nonresponse to phosphodiesterase type 5 inhibitors (PDE5i) is classically attributed to severe end-organ failure with severe tissue alterations, corporeal fibrosis, veno-occlusive dysfunction, and arterial insufficiency [4 6]. In such patients, the use of intracavernous injection of alprostadil represents the second line treatment [7]. In case of nonresponse to an association of alprostatil and PDE5i at the highest doses, the implantation of a penile prosthesis is recommended in patients who wish to recover sexual activity [7,8] because it is unlikely that a recovery of erectile function with or without the use of erectogenic drug might occur [6,9]. We recently showed that patients using alprostadil injections because of insufficient response to sildenafil 1 yr after nerve-sparing RP had no further improvement of erectile function if the same regimen of treatment was 2

3 continued one more year [9]. In the present study, none of the patients reported erection adequate for penetration at baseline with maximal medical treatment as described below. Importantly, all patients declared normal potency before RP with erections hard enough to have satisfactory intercourses without the use of erectogenic drugs. Inclusion criteria: We included men aged years who had had RP 6 mo to 3 yr earlier to treat localized nonaggressive prostate adenocarcinoma without extracapsular extension. Preoperative criteria for nonaggressive cancer were a preoperative prostate-specific antigen (PSA) level <10 ng/ml, a prostate biopsy Gleason score 7, and absence of metastases detected in the preoperative workup; postoperative criteria were an operative specimen Gleason score 7, a localized tumor without extracapsular extension, absence of metastatic lymph nodes, and undetectable PSA. The other inclusion criteria were prp-ed with penile arterial insufficiency and/or venoocclusive dysfunction documented using CDDU as described below, and failure of pharmacotherapy defined as an Erection Hardness Score (EHS) <3 (0, penis does not enlarge; 1, penis is larger but not hard; 2, penis is hard but not hard enough for penetration; 3, penis is hard enough for penetration but not completely hard; and 4, penis is completely hard and fully rigid) after at least 10 intracavernous alprostadil injections (20 µg), combined with sildenafil (100 mg) and the use of a vacuum device, as previously recommended by an international expert panel [10]. Erection hardness is considered a unifying factor for defining response in the treatment of erectile dysfunction [11] and has been recommended to define and report erectile function outcomes after radical prostatectomy [12]. Preparation of BM-MNCs: BM-MNCs include a heterogeneous population of stem cells exhibiting multilineage potential [13,14]. BM-MNCs may secrete soluble factors known to 3

4 contribute to tissue repair via activation of neurogenesis [15], angiogenesis [16], and protection against apoptosis [17]. Bone marrow (BM) aspiration, BM-MMCs preparation, and BM-MNCs injection into both cavernous bodies were performed on the same day in all patients. BM was aspirated from the posterior iliac crest under general anesthesia. The amount collected was 100 ± 15 ml in the two lower dose groups, 417 ± 29 ml in the third dose group, and 782 ± 78 ml in the highest dose group. The BM-MNC fraction was separated using density gradient centrifugation (Ficoll-Paque Premium, Healthcare Bio-Sciences, Sweden), using a manual procedure for the two lower doses and a cell processor (Cobe 2991, Terumo, MD, USA) for the two higher doses in the Etablissement Français du Sang (GMP facility, authorization number: TCG10/R/003) as previously described [18]. The total time needed for cell preparation and administration was less than 5 h. A single syringe containing the BM-MNCs suspended in 60 ml of 4% human albumin (Octapharma, French Licence n ) was prepared and transported to the operating room for administration. Sterility of the injected cell preparation was tested by incubation for at least 7 d of 1% of the total volume of the final product in an automated BacT/ALERT microbial detection system according to the monograph of the European pharmacopeia. Injection: Each BM-MNC suspension was injected into each cavernous body using a 21G needle inserted through the glans after application of a tourniquet at the base of the penis. The injection induced an artificial erection that ensured uniform distribution of the suspension throughout the cavernous bodies. The tourniquet was removed 5 min after the injection. 4

5 Evaluation of BM-MNC on the infused product: The cell counts were performed on a Pentra 60C+ (Horiba Medical, Kyoto, Japan) hematology analyzer from Ficoll-Paque density gradient centrifugation sample. For CD34+ hematopoietic stem cells quantification, samples were diluted to 2x10 7 cells/ml and absolute CD34+ and CD45+ cell numbers and viability (7- aminoactinomycin A) were performed with a Stem Cell Enumeration kit (BD Biosciences): 50 µl of the cell suspension was labeled for 20 min at room temperature with 10 µl of Stem Cell Enumeration reagent and 10 µl of 7-aminoactinomycin A solution, before adding 400 µl of diluted 10 NH 4 Cl solution for red blood cell lysis. A minimum of 75,000 CD45+ gated events were acquired. Acquisition and analysis were performed onto a FacsCanto II flow cytometer with the Facs Diva software (all by BD Biosciences). The frequency of BM-MNC in the infused product was determined by flow cytometric analysis by selecting CD45+ cells with low and intermediate side-scatter events in order to segregate lymphocytes, monocytes and early progenitors and stem cells. For mesenchymal colony-forming unit assay, duplicate aliquots of 2x10 6 cells were seeded in 25-cm 2 tissue-culture flasks containing 5 ml of Dulbecco's Modified Eagle's medium (PAA laboratories, Pasching, Austria) supplemented with 10% fetal bovine serum (Stemcell Technologies, Vancouver, Canada). The culture flasks were placed in a humidified incubator with 5% CO 2 and maintained at 37 C. The growth medium was completely renewed every 3 4 d, and the cultures were stopped on the 10th d. Fibroblast colonies were stained with Giemsa (RAL Diagnostics, Martillac, France) then counted under an inverted microscope at 25 magnification. Aggregates containing more than 50 fibroblasts were classified as colonies. Results were expressed as the mean number of fibroblast colony-forming units per 10 5 mononuclear cells. 5

6 Endpoints: The primary endpoint was the occurrence of BM-MNC therapy-related adverse events within 6 mo after the injection. Body temperature was recorded and pain intensity scored on a 0 10 visual analog scale. Patients were evaluated 1 mo before then 1 mo, 3 mo, and 6 mo after the BM-MNC injection, with special attention to detecting prolonged erection, penile hematoma, local inflammation, or infectious symptoms. A PSA assay and digital rectal examination were performed at each visit to detect cancer recurrence. Hemoglobin level was measured before the procedure and on the postoperative day. The secondary endpoints were sexual function, penile vascularization, and endothelial function, and change in penile length. Sexual function was evaluated at baseline then 1 mo, 3 mo, 6 mo, and 12 mo postinjection, using the EHS [19] and the International Index of Erectile Function-15 (IIEF-15) assessing erectile function, orgasmic function, sexual drive, intercourse satisfaction, and overall satisfaction. Patients could use their preferred erectogenic treatment starting 1 wk after the BM-MNC injection. In order to evaluate natural erections, they were also asked to complete the EHS when not using any treatment. We measured changes in stretched penile length in the flaccid state at each study visit. Penile endothelial function and vascularization were evaluated 6-mo postinjection by performing CDDU using a 5 12 MHz linear array ultrasound probe (PLT-805AT, Toshiba, Tokyo, Japan) as previously described [20]. Endothelial function was assessed using the penile nitric oxide release test (PNORT) by measuring postocclusion changes in cavernous artery diameter [21,22]. The patient was supine with the penis slightly stretched toward the abdomen. The transducer was positioned on the ventral aspect of the penis, and the cavernous arteries evaluated about 3 cm distal to the penoscrotal junction. The diameters of both arteries were recorded. A dedicated sphygmomanometer cuff 2 cm in width was then placed around the base 6

7 of the penis and inflated at 300 mmhg to occlude the arteries. The absence of blood flow beyond the cuff was confirmed using color Doppler. After 3 min with the cuff inflated, the cavernous arteries were evaluated at the same site as before cuff inflation, that is the distal to the cuff. The percent increase in cavernous artery diameter was estimated as Dao-Dbo/Dao, where Dao and Dbo were the mean diameters of the two cavernous arteries after and before occlusion, respectively. PNORT values <35% were taken to indicate endothelial dysfunction [22]. We also measured the peak systolic velocity (PSV), resistive index, and end-diastolic velocity (EDV) in the two cavernosal arteries before and after an intracavernous injection of alprostadil (20 μg). We defined arterial insufficiency as a mean PSV value for both sides <25 cm/s 20 min after the alprostadil injection. Veno-occlusive dysfunction was defined as PSV > 25 cm/s with EDV > 5 cm/s, and resistive index < 0.75 [20]. A greater than 20% difference between the right and left sides was considered to suggest arterial insufficiency, especially when 25 < PSV < 35 cm/s [23]. Statistical analysis: Statistical analyses were performed using Stata v12.1 (StataCorp, College Station, TX, USA). Data are described as mean ± standard deviation or mean ± standard error of the mean, as appropriate. Repeated paired measures at baseline and 1-mo, 3-mo, 6-mo, and 12- mo postinjection were compared using the Wilcoxon signed rank test. Analysis of variance (Fisher test) was performed to assess differences among dose groups at each time point. A p value < 0.05 was considered significant. 7

8 Supplement Results Characteristics of the patient population: All prostate cancers were staged pt2, N0, M0 and all surgical margins were negative. All patients had followed a sexual rehabilitation program including alprostadil injections starting 1 mo after RP and were still using this treatment after a mean of 22.9 ± 9.8 mo since RP. All patients reported a normal erectile function without the use of any erectogenic drug before RP, but they had not been evaluated with specific questionnaires. Sexual scores and CDDU values at baseline are shown in Supplementary Table 1. Adverse events: No modifications of heart rate and blood pressure were observed during the injection of BM-MNCs. No case of prolonged priapism and no cardiovascular events occurred after the procedure. BM aspiration had no impact on white blood cell count and platelet count. Sexual function and response to erectogenic drugs: At 6 mo, nine out of 12 patients were using erectogenic drugs including alprostadil (n = 6), PDE5i (n = 1), combined PDE5i and vacuum (n = 1), and vacuum alone (n = 1). At least one patient in each group was using alprostadil injections at the last follow-up including one patient in group 1, two in group 2, one in group 1, and two in group 4 (ie, three patients of group 1 and group 2 and three patients of group 3 and group 4). Reasons for not using erectogenic drugs in the remaining three patients were as follows: sufficiently hard spontaneous erections for penetration (off-medication EHS, 4) in a dose-3 patient, insufficient response to erectogenic drugs with a request for a penile prosthesis in a dose-1 patient, and loss of the sexual partner shortly after study inclusion with no further sexual activity (EF subscore items 1 4 rated 0) despite improvements in spontaneous erections (off-medication EHS, 0 at baseline and two at 6 mo). Time from RP to BM-MNC injection and RP was not associated with changes in sexual function scores. 8

9 After 6 mo, two patients in the lowest dose group asked for a penile implant because they felt their on-medication erections were unsatisfactory. No technical difficulties related to the previous BM-MNC injection occurred during implantation. Comparing the four dose groups showed no significant differences in sexual function scores (IIEF15 EF subscore and EHS) (Supplementary Fig. 1). Time from RP to BM-MNC injection and RP was not associated with changes in sexual function scores. Sexual function scores at M12 were not significantly different from those at 6 mo in the 10 patients without penile implants (Supplementary Fig. 2), suggesting sustained beneficial effects of BM-MNC injection. Improvements in CDDU parameters: EDV remained unchanged overall, with values remaining >5 cm/s in the eight patients with veno-occlusive dysfunction at baseline. The %PNORT was measured in 11/12 patients in order to assess the effect of BM-MNCs on penile endothelial function. A trend toward an improvement in %PNORT from baseline to 6 mo was noted (p = 0.084). None of the CDDU parameters differed significantly across the four dose groups at any of the time points. BM-MNC characterization: Characteristics of BM-MNCs on the infused product are shown in Supplementary Figure 3 and Supplementary Table 2. All the participants in the clinical study completed the four-dose regimen. 9

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