Prostate cancer (PCa) is the most common noncutaneous cancer in American men, with 1 in 8 men being diagnosed during their lifetime1 and an estimated 3.1 million PCa survivors currently living in the United States.2 Radiotherapy (RT) is a cornerstone of PCa patient care and is indicated as a standard-of-care option, either alone or in combination with surgery and/or hormone therapy, for approximately 60% of patients globally,3,4 although population-based data sets suggest that actual rates of RT vary with time and across risk levels.5 While RT can effectively kill tumor cells, up to 25% of patients experience late bladder toxicities that can persist and are often permanent.6-8 Radiation cystitis (RC) is a debilitating form of dose-limiting toxicity associated with pelvic RT, and while acute RC is generally self-limiting, late RC is often characterized by hematuria that may be severe.9,10 While hyperbaric oxygen therapy can alleviate the symptoms of RC,11,12 there are no Food and Drug Administration-approved preventative treatments of this condition. Efforts to manage other forms of hemorrhagic cystitis such as that which can develop in patients undergoing cyclophosphamide or ifosfamide treatment have largely centered on efforts to mitigate urotoxicity in patients undergoing active treatment or have largely been restricted to positive findings in preclinical models,13 emphasizing the broader challenges associated with preventing cystitis in at-risk patients. A chart review of 709 patients with PCa treated with RT found that 11% developed hemorrhagic cystitis, of whom 52% required blood transfusions.14 A longitudinal survey-based study among PCa survivors found that a decline in urinary function was significantly associated with decisional regret at 6 months and 12 months after treatment,15 and the National Cancer Institute recognizes adverse treatment effects as an important cancer survivorship issue warranting research aimed at reducing the burden of illness and associated costs to the healthcare system.16 Substantial variation in RT responses and associated radiotoxicity are observed in the clinic despite highly conformal RT, and symptom onset is often delayed many years after completion of treatment, highlighting the need to define noninvasive, early biomarkers that can predict the risk of later bladder RC to guide interventional efforts and stratify patients in an effort to mitigate these adverse outcomes.
Extracellular vesicles (EVs) are small membrane-bound particles that are secreted by most cell types and carry macromolecular cargos including proteins, lipids, and nucleotides that can be delivered to specific target cells, thereby influencing physiological and pathological processes.17 These EVs include endolysosomal pathway-derived exosomes and larger microvesicles (MVs) generated by direct membrane budding,18 both of which are highly stable in a range of readily accessible biofluids including serum and urine, providing an opportunity to noninvasively interrogate the characteristics and composition of these vesicles to define biomarkers associated with late RC or other forms of radiotoxicity.19 EV secretion is highly responsive to radiotherapy and other forms of stress, and the functional changes in EV release and composition induced by tumor cell irradiation have been suggested to contribute to the induction of compensatory responses that may support therapeutic resistance and disease progression.20,21 Although other researchers have sought to explore urinary EV-related biomarker profiles in patients with a range of bladder and kidney-related diseases,22 further research is necessary to determine whether changes in EV quantity or cargo content before the onset of late RC in patients with PCa can serve as a biomarker for this clinically important form of late bladder toxicity.
Materials and Methods
Sample Collection
Patient samples were from the University of Rochester Wilmot Cancer Center study, which is a longitudinal, observational study of germline genetics and radiotherapy toxicity (K07 CA187546).23 Participants were recruited between April 2016 and March 2019 from 2 hospitals in the United States (University of Rochester Wilmot Cancer Institute and the Medical College of Wisconsin Froedtert Hospital). Eligible patients were individuals diagnosed with nonmetastatic prostate cancer and scheduled to receive curative-intent external beam radiotherapy with or without brachytherapy boost. Prior prostatectomy and/or hormone therapy was allowed. Urine and blood samples were collected on enrollment (ie, before or on the first day of radiotherapy) and at the end of radiotherapy (last day of fractionated external beam radiotherapy or 1 week after brachytherapy). Clean catch urine was collected in Norgen Biotek 120 cc cups containing a preservative designed for immediate preservation of DNA, RNA, microRNA, and proteins (Norgen Biotek Corp, Ontario, Canada) and stored at room temperature for up to 2 years and then at −80°C for long-term storage, per manufacturer instructions. Serum was isolated from whole blood by centrifugation and stored at −80°C. Participants completed the Expanded Prostate Index Composite24 patient-reported outcome (PRO) questionnaire supplemented with questions on radiation-related symptoms and quality of life at the same time that urine and serum were collected as well as 6 months and annually after radiotherapy, up to 5 years. Demographic, clinical, and treatment data were abstracted from electronic medical records. Gross hematuria was determined at each assessment using the PRO questionnaire, as previously described.23 A set of approximately 2 patients without hematuria and comparable follow-up was randomly selected for each hematuria case available.
Nanoparticle Tracking Analysis
Urine and serum samples were thawed at room temperature, centrifuged twice at 15,000 ×g to remove cellular debris, and transferred into Eppendorf tubes. Samples were then analyzed using a Nanosight NS3000 instrument (Malvern Panalytical, UK). Urine samples were diluted 1:2 to 1:128 in Dulbecco phosphate buffered saline (DPBS; Gibco) as necessary to avoid the oversaturation of the captured images while serum samples were diluted 1:80 to 1:1000 as appropriate. Particle data for each sample were collected in one 60-second video file, with microfluidics being used to constantly flow samples over the imaging stage during this capture process. Camera level and detection threshold values remained constant for all analyses to ensure consistency. Data were collected by investigators blinded to patient hematuria status.
Statistical Analysis
All analyses and figure generation were performed using GraphPad Prism 9.2 (GraphPad Software LLC). Results are presented as mean with SEM. Data were compared between 2 groups using Student t-tests or Mann-Whitney U tests or between multiple groups using 2-way analyses of variance (ANOVAs) with the Tukey multiple comparison test. P < .05 was established as the threshold of significance for these analyses.
Results
To explore the potential utility of EVs as early biomarkers of late RC and other forms of RT-induced bladder toxicity, we analyzed the EV profiles of preserved urine and serum samples previously collected from 30 patients with PCa undergoing standard-of-care pelvic RT as part of a longitudinal study of late radiotoxicity.23 This cohort included 9 patients who ultimately developed gross hematuria after RT and 21 patients who did not develop hematuria after similar radiation treatment and follow-up (Table 1). These 2 patient groups were similar in key demographic, clinical, and treatment variables.
Table 1. -
Participant Demographic Characteristics
|
All patients
N=30 |
With Post-RT Hematuria N=9 |
Without Post-RT Hematuria
N=21 |
P value
a
|
| Age at radiotherapy, median (range), years |
70 (55-86) |
75 (63-86) |
69 (55-86) |
0.08 |
| BMI, median (range) |
29.5 (21.4-40.1) |
28.5 (21.4-39.9) |
29.9 (21.8-40.1) |
0.54 |
| Smoking status, N (%) |
|
|
|
0.46 |
| Never |
18 (60.0) |
7 (77.8) |
11 (52.4) |
|
| Former |
10 (33.3) |
2 (22.2) |
8 (38.1) |
|
| Current |
2 (6.7) |
0 |
2 (9.5) |
|
| Diabetes |
|
|
|
0.64 |
| No |
24 (80.0) |
8 (88.9) |
16 (76.2) |
|
| Yes |
6 (20.0) |
1 (11.1) |
5 (23.8) |
|
| Heart disease |
|
|
|
0.62 |
| No |
25 (83.3) |
7 (77.8) |
18 (85.7) |
|
| Yes |
5 (16.7) |
2 (22.2) |
3 (14.3) |
|
| Hypertension |
|
|
|
1.00 |
| No |
11 (36.7) |
3 (33.3) |
8 (38.1) |
|
| Yes |
19 (63.3) |
6 (66.7) |
13 (61.9) |
|
| Taking an ACE inhibitor |
|
|
|
0.25 |
| No |
18 (60.0) |
7 (77.8) |
11 (52.4) |
|
| Yes |
12 (40.0) |
2 (22.2) |
10 (47.6) |
|
| Tumor stage |
|
|
|
0.19 |
| T1 |
16 (53.3) |
5 (55.6) |
11 (52.4) |
|
| T2 |
8 (26.7) |
3 (33.3) |
5 (23.8) |
|
| T3 or T4 |
6 (20.0) |
1 (11.1) |
5 (23.8) |
|
| Gleason score |
|
|
|
0.38 |
| ≤6 |
5 (16.7) |
3 (33.3) |
2 (9.5) |
|
| 7 |
15 (50.0) |
4 (44.4) |
11 (52.4) |
|
| ≥8 |
10 (33.3) |
2 (22.2) |
8 (38.1) |
|
| National Comprehensive Cancer Network risk group |
|
|
|
0.73 |
| Low |
4 (13.3) |
2 (22.2) |
2 (9.5) |
|
| Intermediate |
19 (63.3) |
5 (55.6) |
14 (66.7) |
|
| High or very high |
7 (23.3) |
2 (22.2) |
5 (23.8) |
|
| Prior prostatectomy |
|
|
|
0.07 |
| No |
21 (77.8) |
9 (100) |
12 (66.7) |
|
| Yes |
6 (22.2) |
0 |
6 (33.3) |
|
| Prior transurethral prostate or bladder resection |
|
|
|
0.14 |
| No |
25 (83.3) |
6 (66.7) |
19 (90.5) |
|
| Yes |
5 (16.7) |
3 (33.3) |
2 (9.5) |
|
| Androgen deprivation therapy |
|
|
|
0.39 |
| No |
9 (30.0) |
4 (44.4) |
5 (23.8) |
|
| Yes |
21 (70.0) |
5 (55.6) |
16 (76.2) |
|
| Brachytherapy boost |
|
|
|
0.62 |
| No |
25 (83.3) |
7 (77.8) |
18 (85.7) |
|
| Yes |
5 (16.7) |
2 (22.2) |
3 (14.3) |
|
| Bladder volume (%) receiving 70 Gy, median (range) |
6.7 (0-30.3) |
12.1 (0-30.3) |
6.5 (0-27.2) |
0.48 |
ACE, angiotensin-converting enzyme; RT, radiotherapy.
aFor continuous variables, P-values are derived from Wilcoxon rank-sum tests; for categorical variables, P-values are derived from chi-square tests or when fewer than 5 samples were included per cell, Fisher exact test
A nanoparticle tracking analysis (NTA) approach was used to analyze the number of EVs in matched pre-RT and post-RT samples from these patients. Longitudinal sample collection revealed that urine of patients with PCa who ultimately developed hematuria contained significantly higher EV concentrations after RT treatment relative to those before RT treatment, whereas no RT-related changes in urinary EV counts were evident in patients who did not develop hematuria (Figures 1A and 1B). Because blood samples are routinely collected from patients with cancer undergoing treatment and have the potential to reflect systemic RT-related tissue damage and toxicity risks, EV concentrations in serum samples from these same patients with PCa were also measured. Although patients who did develop hematuria exhibited a statistically significant increase in EV concentrations relative to their pretreatment levels (Figure 1C), these differences were far more modest than those observed for urine samples with a mean 1.686-fold change as compared with a median 4.32-fold change for urine samples. Moreover, no differences in absolute particle counts were evident among samples (Figure 1D).
;)
Figure 1.: Post-RT hematuria is associated with elevated post-treatment extracellular vesicle counts in PCa patient urine and serum. Urine and serum samples from patients with PCa undergoing treatment were collected before and after RT. Extracellular vesicles in these samples were measured by nanoparticle tracking analysis. A, Fold-change in total urine EVs for each patient (post-RT/pre-RT). B, Total urine EVs. C, Fold-change in total serum EVs for each patient. D, Total serum EVs. (E-H) Patient urine sample data were analyzed to assess the numbers of exosomes (diameter: 30-100 nm) (E, F) and microvesicles (diameter: 100-300 nm) (G, H) in these samples. (I-L) Patient serum sample data were analyzed to assess the numbers of exosomes (I, J) and microvesicles (K, L) in these samples. Patients are grouped according to whether they did or did not develop post-RT hematuria (red and black, respectively). Data are presented as mean ± SEM and were compared using Student t-tests or Mann-Whitney U tests (2 groups) or 2-way ANOVAs with the Tukey multiple comparison test (more than 2 groups).*P < .05, **P < .01, ***P < .001, ****P < .0001. Statistical comparisons not shown were not significant (ns). EV, extracellular vesicle; PCa, prostate cancer; RT, radiotherapy.
To extend these analyses and further explore the predictive value of different EV subpopulations as biomarkers of RT-associated late bladder toxicity in patients with PCa, the numbers of exosomes (diameter: 30-100 nm) and MVs (diameter: 100-300 nm) in these samples were measured using NTA. Strikingly, urine samples from patients with hematuria exhibited significantly higher post-RT concentrations of both exosomes (Figures 1E and 1F) and MVs (Figures 1G and 1H), particularly when assessing fold-change values. By contrast, serum samples from patients with hematuria exhibited a significant increase in post-RT exosome concentrations (Figures 1I and 1J), whereas MV concentrations were unchanged with hematuria status (Figures 1K and 1L).
Discussion
Prostate cancer is one of the most prevalent forms of cancer in men, and RT remains the standard of care for most patients with PCa in the United States. As treatment options improve and survivorship rates rise, however, the morbidity associated with RT-associated late bladder toxicity, which can cause long-term and potentially permanent damage,6-8 represents an increasingly important focus for clinical care and research. These adverse toxicities can be disruptive and, in severe cases, may require patients to undergo further invasive procedures with their attendant risks.10,11 Given that late RC may manifest anywhere from 6 months to 20 years after pelvic RT exposure,9,11 the ability to predict the risk of late RC or other forms of late bladder toxicity in patients before such radiotoxicity manifests may thus provide an opportunity for interventional efforts that can better protect patients. These results suggest that increases in EV concentrations in post-RT urine, and to a lesser extent post-RT serum, of patients with PCa can serve as an early predictor of future hematuria development many months or years after RT. Urinary EVs may thus represent a powerful, inexpensive, and noninvasive prognostic biomarker associated with the risk of late bladder toxicity that can guide clinical interventions to preserve bladder integrity and function.
EVs are a heterogeneous population of lipid-enclosed nanovesicles released by most cell types that include exosomes generated by the endolysosomal pathway through Rab27α-mediated mechanisms and larger microvesicles that bud directly from the cell membrane.18 Cancer-related changes in EV composition have led to widespread interest in their value as disease-related biomarkers19; various oncogenic and proto-oncogenic proteins have been detected as cargos within cancer EVs. Works from our lab has shown that urinary EVs derived from bladder cancer patients and cells contain diverse bioactive cargo proteins, including EGF-like repeats and discoidin domains 3,25 and the levels of these urinary EV cargo proteins are correlated with worse cancer-specific survival in bladder cancer patients.26 Biofluid-derived EVs are also functionally active mediators of cell-cell communication that can regulate oncogenesis27 and modulate tumor cell resistance to radiotherapy and other interventions.20 Accordingly, further research is warranted to characterize the cargo content within post-RT EVs from patients with PCa who do and do not develop hematuria to clarify the functional role of these vesicles, with a particular focus on whether exosomes and microvesicles play nonredundant roles given the observed differences in the predictive power of these EV subpopulations. The cargo profiles in these EVs may also reflect the status of the cells from which they are derived and the cellular processes underlying their biogenesis, potentially highlighting viable targets for further interventional research aimed at mitigating post-RT toxicity in patients undergoing RT. Although the NTA-based approach to EV quantification used in this study is considered to be a reliable and stable assay, it may be a relatively low-throughput strategy for large-scale clinical application. Many laboratories and companies are actively developing techniques and technologies that may improve the feasibility of incorporating EVs into standard clinical workflows for appropriate patients, as reviewed previously.28,29
Despite its potential severity, the etiology of late RC and associated hematuria after RT treatment in patients with PCa remains under-researched and incompletely understood owing to its heterogeneous presentation, multifactorial etiology, and complex biology. In the largest genome-wide association study (GWAS) of late bladder radiotoxicity to date (n = 3,871 patients), several candidate causal single nucleotide polymorphisms (SNPs) were found to be significantly associated with RT-induced hematuria including SNPs in the AGT gene, which encodes angiotensinogen.30 Angiotensin-converting enzyme inhibitors (ACEis) have been shown to effectively mitigate various radiation injuries including proctitis31 in patients with PCa, and analysis of ACEis among the same patient cohort enrolled in this study confirmed a strong protective effect against late hematuria.23 Strikingly, 2 of the top 10 protein-coding genes most closely associated with the development of hematuria in this GWAS analysis were SYTL3 (synaptotagmin like 3), which encodes a Rab27α effector protein,32 and COG2 (component of oigometric Golgi complex 2), which encodes a vesicle docking protein.33 The close relationship between these 2 hematuria-related genes and membrane vesicle trafficking suggests a functional link between EV biogenesis and RT-induced late hematuria, strengthening the findings of this study while emphasizing the need to further examine how EVs and EV-related secretory pathways contribute to bladder toxicity. Whether interventional efforts targeting EV biogenesis-related pathways have the potential to mitigate the pathogenesis of hematuria remains to be determined. Exploring whether EV induction can similarly predict patient susceptibility to developing hemorrhagic cystitis in response to treatment with cyclophosphamide or related drugs13 is another promising avenue for further investigation, particularly because there is limited preclinical evidence that cyclophosphamide can provoke increased EV release from certain cancer cell lines in vitro.34 Given that EVs can also play complex functional roles in a range of other inflammatory settings,35,36 the degree to which urinary EV concentrations and composition correlate with inflammatory phenotypes more generally or predict functional outcomes in other contexts also warrants additional study.
In this study, within-individual longitudinal EV comparisons were most closely associated with hematuria status, but pretreatment EV concentrations were also relatively stable and normally distributed in urine samples (109-1010 particles/mL). Such stability makes these urinary EV counts an even more promising biomarker for post-RT patient monitoring given that it enables the potential detection of aberrantly elevated post-RT EV concentrations even when pretreatment samples are unavailable.
Because the observed increase in urinary and serum EV abundance in patients who ultimately developed hematuria was only evident after the completion of RT treatment when RT dose modification is no longer possible, interventional strategies that can preemptively mitigate subsequent RC development will be necessary to optimally leverage this clinical biomarker to improve patient outcomes. Unfortunately, Food and Drug Administration-approved treatments capable of mediating such protection are lacking at present, highlighting an important area of unmet clinical need that warrants further study. Although hyperbaric oxygen therapy is deployed as a treatment of RC-related symptoms,11,12 there is no evidence indicating that it can prevent the onset of such damage after RT is complete. The significant reduction in late bladder toxicity observed in patients with PCa undergoing ACEi treatment at the start of RT23 and our preclinical RT animal model37 also highlights the opportunity to explore the post-RT benefits of ACEi administration, and building on the findings associated with the prevention and management of cyclophosphamide-induced hemorrhagic cystitis may also inform these efforts.13 Whether these or other treatment strategies have any impact on urinary or serum EV profiles also remains to be investigated, with such research having the potential to offer further insight into the functional roles that EVs play as mediators of post-RT tissue damage.
Conclusion
Post-treatment EV concentrations in urine samples collected from patients with PCa undergoing RT are associated with the future onset of hematuria, providing a promising biomarker that may enable the more timely treatment of at-risk patients to mitigate the development of late radiation cystitis in any individuals undergoing pelvic RT. Serum samples may also offer similar albeit less robust prognostic utility, particularly when specifically focusing on exosome populations. These data highlight the need for more detailed studies of EV kinetics, characteristics, and composition to gain insight into the mechanisms governing normal tissue toxicity in patients undergoing the RT-based treatment of a range of cancers while also underscoring an unmet clinical need for the development of therapies that protect against bladder damage in individuals identified as being at risk. Future prospective validation of our findings in a larger clinical study will be critical, as will efforts to determine whether the EVs present in post-RT patient samples play a functional role in shaping the risk of hematuria and other forms of delayed bladder toxicity.
Authors' Contributions
Y.F.L., R.D.M., and S.L.K. conceived and designed the study. B.M. and Y.C. contributed important design considerations. R.D.M. and E.O. purified urinary and serum EVs, performed experiments, and conducted statistical analyses. R.D.M. generated the figures and wrote the manuscript. All authors interpreted the data and critically revised the final manuscript.
Ethics Approval and Consent to Participate
Collection of urine and tissue was approved by the University of Rochester Research Subjects Review Board. Written informed consent was received from participants prior to inclusion in the study. All aspects of the study were performed in accordance with the Declaration of Helsinki.
Competing Interests
The authors declare no conflict of interest.
Funding Information
This work is supported by the Urology Departmental Research fund.
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