18F-DCFPyL (PSMA) PET as a radiotherapy response assessment tool in metastatic prostate cancer

Abstract

Background:
Prostate Specific Membrane Antigen (PSMA) - positron emission tomography (PET) guides metastasis-directed radiotherapy (MDRT) in prostate cancer (PrCa). However, its value as a treatment response assessment tool after MDRT remains unclear. Importantly, there is limited understanding of the potential of radiotherapy (RT) to alter PSMA gene (folate hydrolase 1; FOLH1) expression.

Methodology:
We reviewed a series of 11 men with oligo-metastatic PrCa (25 metastasis sites) treated with MDRT before re-staging with 18F-DCFPyL (PSMA) PET upon secondary recurrence. Acute effects of RT on PSMA protein and mRNA levels were examined with qPCR and immunoblotting in human wild-type androgen-sensitive (LNCap), castrate-resistant (22RV1) and castrate-resistant neuroendocrine (PC3 and DU145) PrCa cell lines. Xenograft tumors were analyzed with immunohistochemistry. Further, we examined PSMA expression in untreated and irradiated radio-resistant (RR) 22RV1 (22RV1-RR) and DU145 (DU145-RR) cells and xenografts selected for survival after high-dose RT.

Results:
The majority of MDRT-treated lesions showed lack of PSMA-PET/CT avidity, suggesting treatment response even after low biological effective dose (BED) MDRT. We observed similar high degree of heterogeneity of PSMA expression in both human specimens and in xenograft tumors. PSMA was highly expressed in LNCap and 22RV1 cells and tumors but not in the neuroendocrine PC3 and DU145 models. Single fraction RT caused detectable reduction in PSMA protein but not in mRNA levels in LNCap cells and did not significantly alter PSMA protein or mRNA levels in tissue culture or xenografts of the other cell lines. However, radio-resistant 22RV1-RR cells and tumors demonstrated marked decrease of PSMA transcript and protein expression over their parental counterparts.

Conclusions:
PSMA-PET may be a promising tool to assess RT response in oligo-metastatic PrCa. However, future systematic investigation of this concept should recognize the high degree of heterogeneity of PSMA expression within prostate tumors and the risk for loss of PSMA expression in tumor surviving curative courses of RT.

Full text

Clinical and Translational Radiation Oncology 39 (2023) 100583
Available online 18 January 2023
2405-6308/© 2023 The Author(s). Published by Elsevier B.V. on behalf of European Society for Radiotherapy and Oncology. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
18
F-DCFPyL (PSMA) PET as a radiotherapy response assessment tool in
metastatic prostate cancer
Aruz Mesci
a
,
d
, Elham Ahmadi
d
,
h
, Amr Ali
d
,
h
, Mohammad Gouran-Savadkoohi
a
,
d
,
Evangelia Evelyn Tsakiridis
e
,
h
, Olga-Demetra Biziotis
d
,
h
, Tom Chow
b
, Anil Kapoor
c
,
Monalisa Sur
f
, Gregory R. Steinberg
e
,
h
, Stanley Liu
i
, Katherine Zukotynski
g
,
Theodoros Tsakiridis
a
,
d
,
h
,
*
a
Radiation Oncology, Juravinski Cancer Centre, Hamilton Health Sciences, Ontario, Canada
b
Physics, Juravinski Cancer Centre, Hamilton Health Sciences, Ontario, Canada
c
Division of Urology, McMaster University and St. Joseph’s Hospital, Hamilton, Ontario, Canada
d
Dept. of Oncology, McMaster University, Hamilton, Ontario, Canada
e
Dept. of Medicine, McMaster University, Hamilton, Ontario, Canada
f
Dept. of Pathology, McMaster University, Hamilton, Ontario, Canada
g
Dept. of Radiology, McMaster University, Hamilton, Ontario, Canada
h
Centre for Metabolism, Obesity and Diabetes Research, McMaster University, Hamilton, Ontario, Canada
i
Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Ontario, Canada
ARTICLE INFO
Keywords:
PSMA-PET
FOLH1 expression
Immunohistochemistry
qPCR
Radio-resistance
Tumor heterogeneity
ABSTRACT
Background: Prostate Specic Membrane Antigen (PSMA) – positron emission tomography (PET) guides
metastasis-directed radiotherapy (MDRT) in prostate cancer (PrCa). However, its value as a treatment response
assessment tool after MDRT remains unclear. Importantly, there is limited understanding of the potential of
radiotherapy (RT) to alter PSMA gene (folate hydrolase 1; FOLH1) expression.
Methodology: We reviewed a series of 11 men with oligo-metastatic PrCa (25 metastasis sites) treated with MDRT
before re-staging with
18
F-DCFPyL (PSMA) PET upon secondary recurrence. Acute effects of RT on PSMA protein
and mRNA levels were examined with qPCR and immunoblotting in human wild-type androgen-sensitive
(LNCap), castrate-resistant (22RV1) and castrate-resistant neuroendocrine (PC3 and DU145) PrCa cell lines.
Xenograft tumors were analyzed with immunohistochemistry. Further, we examined PSMA expression in un-
treated and irradiated radio-resistant (RR) 22RV1 (22RV1-RR) and DU145 (DU145-RR) cells and xenografts
selected for survival after high-dose RT.
Results: The majority of MDRT-treated lesions showed lack of PSMA-PET/CT avidity, suggesting treatment
response even after low biological effective dose (BED) MDRT. We observed similar high degree of heterogeneity
of PSMA expression in both human specimens and in xenograft tumors. PSMA was highly expressed in LNCap
and 22RV1 cells and tumors but not in the neuroendocrine PC3 and DU145 models. Single fraction RT caused
detectable reduction in PSMA protein but not in mRNA levels in LNCap cells and did not signicantly alter PSMA
protein or mRNA levels in tissue culture or xenografts of the other cell lines. However, radio-resistant 22RV1-RR
cells and tumors demonstrated marked decrease of PSMA transcript and protein expression over their parental
counterparts.
Abbreviations: ADT, Androgen Deprivation Therapy; AMACR, Alpha-Methylacyl-CoA Racemase; ARAT, Androgen Receptor Axis-Targeted; BED, Biological
Effective Dose; CRPC, Castration Resistant Prostate Cancer; FOLH1, Folate Hydrolase 1; H&E, Hematoxylin and Eosin; H-Score, Histologic Score; HSPC, Hormone
Sensitive Prostate Cancer; IHC, Immunohistochemistry; LHRH, Luteinizing Hormone Releasing Hormone; mCRPC, Metastatic Castration Resistant Prostate Cancer;
MDRT, Metastasis Directed Radiotherapy; mRNA, Messenger Ribonucleic Acid; NH, Hormone Naïve; PET, Positron Emission Tomography; P-H3, Phosphorylated
Histone-H3; PrCa, Prostate Cancer; PSA, Prostate Specic Antigen; PSMA, Prostate Specic Membrane Antigen; qPCR, Quantitative Polymerase Chain Reaction; Rec,
Recurrence; RP, Radical Prostatectomy; RT, Radiation Therapy; SUV, Standardized Uptake Value.
* Corresponding author at: Radiation Oncologist, Juravinski Cancer Center, Dept. of Oncology, McMaster University, Hamilton, ON L8V 5C2, Canada.
E-mail address: theos.tsakiridis@mcmaster.ca (T. Tsakiridis).
Contents lists available at ScienceDirect
Clinical and Translational Radiation Oncology
journal homepage: www.sciencedirect.com/journal/clinical-and-translational-radiation-oncology
https://doi.org/10.1016/j.ctro.2023.100583
Received 17 October 2022; Received in revised form 13 January 2023; Accepted 15 January 2023

Clinical and Translational Radiation Oncology 39 (2023) 100583
2
Conclusions: PSMA-PET may be a promising tool to assess RT response in oligo-metastatic PrCa. However, future
systematic investigation of this concept should recognize the high degree of heterogeneity of PSMA expression
within prostate tumors and the risk for loss of PSMA expression in tumor surviving curative courses of RT.
Introduction
Positron emission tomography (PET) using low-molecular-weight
ligands of prostate-specic membrane antigen (PSMA) provides signif-
icant improvements in prostate cancer (PrCa) detection over conven-
tional imaging with computed tomography (CT) and bone scan (BS) [1].
Studies utilizing PSMA-PET in the initial staging or at biochemical
failure have demonstrated superior sensitivity and specicity compared
to conventional imaging [2–6]. PSMA is the product of folate hydrolase
1 (FOLH1) gene, a type II transmembrane protein expressed in prostate,
kidney, small intestine, as well central and peripheral nervous system
[7]. PSMA is suggested to have metabolic roles in the nervous and
gastrointestinal systems [8,9]. Studies showed that PSMA expression is
higher in malignant prostate glands compared to normal cells [10];
however, potential contribution of PSMA to PrCa oncogenesis is unclear.
Nonetheless, development of neuroendocrine features in PrCa, an
aggressive state with poor prognosis [11], is associated inversely with
PSMA expression [12].
PSMA expression was investigated in a limited number of studies,
which showed signicant heterogeneity in its expression, raising ques-
tions about its reliability as a disease marker. Most reports showed
PSMA staining in most PrCa patients [13,14]; however, variation
amongst patients is signicant. Further, Mannweiler et al. [14] found
discordance in the percentage of the tumour expressing PSMA between
primary prostate tumor and metastatic tissues from the same patients.
The proportion of tumor expressing PSMA appears to have clinical
consequences, as Ferraro et al. [13] found that high percentage of
PSMA-negative tumour regions correlated strongly with the probability
of negative PSMA-PET, despite high PSA values.
Today, PSMA-PET is regarded as the preferred imaging modality for
the detection of recurrent disease, but its specic utility in response
assessment after local therapy is less clear. Few reports address PSMA
kinetics post radiotherapy. In a small case series of 5 oligo-metastatic
PrCa patients treated with metastasis-directed RT (MDRT), high rates
of SUV-based response were recorded [15]. Of 18 lesions reported, only
2 showed progression. Despite the small sample size, the authors noted a
relationship between decrease in SUVmax and time interval between RT
and PSMA.
Regulation of PSMA expression is not well understood. Early pre-
clinical studies showed that treatment of PrCa cell lines with andro-
gens suppresses PSMA expression in an androgen receptor-dependent
manner [16]. Concordantly, in vitro expression of PSMA was increased
in response to commonly used androgen-receptor axis-targeted treat-
ments such as enzalutamide [16] and abiraterone [17,18]. The potential
of cytotoxic therapy to regulate PSMA expression had not been inves-
tigated until recently. While this study was prepared for publication,
Sheehan et al. [19] reported evidence that PSMA expression can be
upregulated by some DNA damaging agents, including topoisomerase-2
inhibitors (daunorubicin) and ionizing radiation. They detected upre-
gulation of PSMA protein expression in castration-sensitive LNCap cells
and castration-resistant LNCap95 and 22RV1 cells and PDX tumor
models by low BED irradiation but no signicant change at the mRNA
(FOLH1).
In this report, we show a series of 11 patients who received RT to
metastatic lesions (MDRT) detected using either
18
F-DCFPyL (PSMA)
PET and/or conventional imaging followed by PSMA-PET after sec-
ondary biochemical progression. Elimination of PET avidity in the ma-
jority of cases suggests either, i) a high rate of metastatic PrCa response
to RT (supported by the associated CT ndings), and/or ii) that RT could
potentially down-regulate PSMA/FOLH1 levels in treated tissues. Given
the signicant challenge of obtaining tissue from sites of metastatic
disease, we pursued a preclinical analysis of the regulation of FOLH1/
PSMA expression by RT. FOLH1/PSMA transcript and protein levels
were examined in established human parental and radio-resistant PrCa
cell lines and xenografts after RT to elucidate the effects of RT at the
cellular and tumor level.
Methods
Patients
Eleven patients with oligo-metastatic PrCa (ve or less metastatic
sites) participating in a Hamilton Integrated Health Research Board
(HiREB)-approved PSMA-PET registry were included in this study. Pa-
tients were treated with MDRT to one or more oligometastatic lesions,
and subsequent biochemical failure had imaging with
18
F-DCFPyL-PET
performed 5–72 months after radiotherapy (Table 1).
18
F-DCFPyL (333
MBq [9 mCi]) was administered intravenously 60+/-10 min before
imaging. PET/CT was performed using a 64-slice Discovery RX scanner
(GE Healthcare). All cases reported in this study had their baseline
conventional / PET imaging and post-MDRT PSMA-PET scans reviewed
by two nuclear medicine experts (the reporting physician and Dr. KZ co-
author). For each PET/CT study acquired post-radiation therapy, each
site of irradiated disease was evaluated for
18
F-DCFPyL avidity (SUV-
max). In addition, the PET/CT scans were compared with conventional
imaging available prior to MDRT. In patients who underwent both
baseline and secondary or tertiary (2nd/3rd) recurrence PSMA-PET/CT
around MDRT, the change in avidity and morphology of each radiated
lesion between the PET studies was determined by both CT and PET
images and the presence of new lesions was documented.
Cells
Wildtype LNCap, PC3, 22RV1 and DU145 PrCa cell lines were pur-
chased from ATCC. Radiation-resistant 22RV1-RR and DU145-RR were
generated by serially treating wild-type DU145 and 22RV1 cells with 2
Gy daily fractions (Monday-Friday) to a total of 118 Gy, as described
[20]. Treatments: Cells were grown in culture to 70–80 % conuence,
were irradiated (RT) 0, 2 or 8 Gy with a clinical linear accelerator and
parallel-opposed beams (6MV), using established methodology (as
described [21]), followed by incubation for 24 or 48 h post-treatment for
qPCR and immunoblotting experiments, respectively.
Xenografts
Using protocols approved by the McMaster University Animal
Research Ethics Board, cells (1 ×10
6
/100
μ
L), suspended in 50:50
mixture of ice-cold Matrigel: phosphate, were injected subcutaneously
into the anks of 8-week old male BALB/c nude mice (CAnN.Cg-
Foxnnu/Crl; Charles River: Wilmington, MA). Growth kinetics were
monitored using a caliper and tumor volume was calculated using
V=½(length ×width
2
). When tumors reached 100 mm
3
, mice were
randomly assigned to control or RT (5 Gy), delivered by a clinical linear
accelerator and parallel-opposed beams (6MV; equally weighted left and
right lateral) using established methodology, as described [21]. Tumor
progression was monitored until each tumor reached endpoint of 2200
mm
3
. Extracted tumors were bisected and were either formalin-xed
parafn-embedded (FFPE) or snap frozen with liquid N
2
.
A. Mesci et al.

Clinical and Translational Radiation Oncology 39 (2023) 100583
3
Table 1
Cohort of patients treated with MDRT and subsequent PSMA-PET. Ages at rst MDRT, initial diagnoses, site(s) of metastases, associated MDRT dose/fractionation
(and BED
1.5
), concurrent systemic therapy, interval between MDRT and PSMA-PET scan (in months, for each course), interpretation of response based on PSMA-PET,
overall pattern of recurrence at PSMA, and subsequent management synopsis are given.
Patient
Number
(Age at
MDRT:
years)
Initial Diagnosis
(Dx) and treatment
[Dx before initial
MDRT]
(Dx at further
progression)
Sites of Metastases Radiotherapy
(MDRT)
Regimen
(BED
1.5
)
Concurrent
Systemic
therapy
at time of PSMA-
PET
Time from
RT to PSMA
(in mo)
Response
(SUVmax)
Order of Imaging modalities
PSMA Findings
[1st line metastasis
management]
(2nd line metastasis
management])
1 (68) mCRPC,
GG5, PSA 12, cT3b
ADT +ARAT
[mCRPC]
1. LUL nodule
2. LUL nodule
3. LLL nodule
1. 48 Gy/4 (432)
2. 48 Gy/4 (432)
3. 48 Gy/4 (43
2)
Yes
ADT +ARAT
7.6
7.6
7.6
1. No (8.4)
2. No (4.4)
3. Partial
(5.3)
Baseline BS/CT &:
[MDRT]
2nd Rec. PSMA-PET &:
Oligo-metastatic recurrence;
(MDRT)
2 (71) mHSPC,
GG4, PSA 40, cT3b
Pelvic RT +ADT
[mHSPC]
4. T5 vertebra 4. 20 Gy/5 (73) No 72.2 4. Yes (*) Baseline BS/CT:
[MDRT +ADT ×36mo]
2nd Rec PSMA-PET:
Mixed local prostate, regional
and oligometastatic recurrence;
(MDRT +ADT)
3 (66) Localized PrCa
GG3, PSA 5.5, pT3a,
RP: positive margin
Salvage RT
[mHSPC]
5. T7 vertebra
6. L 3rd rib
7. S1 vertebra**
8. R-external iliac
LN**
9. R-iliac ala**
5. 20 Gy/5 (73)
6. 20 Gy/5 (73)
7. 30 Gy/5 (150)
8. 30 Gy/5(1 5
0)
9. 30 Gy/5(150)
No 17.9
47.3
27
27
27
5. Yes[2nd]/
No[3rd]
$
6. Yes[2nd]/
Yes[3rd]
7. Yes[3rd]
(*)
8. Yes[3rd]
(*)
9. Yes[3rd]
(*)
Baseline CT/BS
[MDRT]
2nd Rec. PSMA-PET:
Oligo-metastatic recurrence,
[MDRT]
3rd Rec. PSMA-PET: Oligo-
metastatic recurrence $ (MDRT,
ADT +MDRT)
4 (77) Localized PrCa
GG3, PSA 10, pT2b
RP, Salvage RT
[HN-mHSPC]
10. L-clavicle 10. 35 Gy/5
(198)
No 21.2 10. Yes (*) Baseline BS-CT:
[MDRT]
2nd Rec. PSMA-PET:
Poly-metastatic recurrence;
(palliative RT and ADT)
5 (69) Localized PrCa,
GG3, PSA 7.6, cT2b
RT
[HN-HSPC]
(mHSPC)
(mCRPC)
11. R iliac bone
12. R pubic bone
13. L3 vertebra
11. 30 Gy/10
(90)
12. 30 Gy/10
(90)
13. 16 Gy/1
(187)
No 11.4
11.4
11.4
11. Yes (*)
12. Yes (*)
13. No (18.3)
Baseline BS/CT:
[MDRT]
2nd. Rec. PSMA-PET: Oligo-
metastatic recurrence.
Later: Poly-metastatic recurrence
(MDRT, ADT +ARAT)
6 (69) Localized PrCa
GG3, PSA 7.4
RT
[HN-HSPC]
(mHSPC)
14. Perirectal LN
15. Subcarinal LN
16. L hilar LN
17. L iliac bone
14. 30 Gy/5
(150)
15. 30 Gy/5
(150)
16. 30 Gy/5
(150)
17. 30 Gy/5
(150)
No 8.4
8.4
8.4
8.4
14. Yes (3.2)
15. Yes (4.8)
16. Yes (4.8)
17. Yes (2.8)
Baseline BS/CT and PSMA-PET:
Oligometastatic recurrence;
[MDRT]
2nd Rec. PSMA-PET:
Poly-metastatic recurrence;
(MDRT, ADT +ARAT)
7 (65) Localized PrCa
GG5, PSA 51,
RT +ADT
[HN-HSPC]
(mHSPC)
18. Para-aortic LN 18. 35 Gy/5
(198)
No 15.5 18. Yes (4.4) Baseline BS/CT and PSMA-PET:
Oligometastatic recurrence;
[MDRT]
2nd Rec. PSMA-PET: Poly-
metastatic recurrence
8 (69) Localized PrCa
GG2, PSA 11.2, cT2c
RP, Salvage RT,
[CRPC]
(mCRPC)
19. R 9th rib
20. R SI joint
19. 35 Gy/5 (19
8)
20. 35 Gy/5
(198)
Yes
ADT+
ARAT
6.2
6.2
19. Yes (*)
20. Yes (*)
Baseline BS/CT &:
[MDRT]
2nd Rec. PSMA-PET&: Poly-
metastatic recurrence conned to
paraaortic nodes;
(MDRT)
9 (74) Localized PrCa
GG3, PSA 12.7, pT3a
RP, Salvage RT
[CRPC]
(mCRPC)
21. L2-3 vertebra
22. T3 vertebra
(**)
23. L3 vertebra
(re-treat, **)
21. 20 Gy/5 (73)
22. 24 Gy/2
(216)
23. 18 Gy/2
(126)
Yes
ADT+
ARAT
5.3
5.6
5.6
21. No (11.6)
22. No (19.3)
23. Partial
(9.6)
Baseline BS/CT &:
2nd Rec. PSMA-PET &:
Oligometastatic recurrence.
3rd Rec. PSMA-PET &:
Oligometastatic recurrence;
(repeat MDRT)
10 (70) mHSPC,
GG2, PSA 178, cT2b
MDRT +ADT
[mHSPC]
24. R-inferior
pubic ramus
24. 40 Gy/20 ADT 23.6 24. Yes (*) Baseline BS/CT:
[MDRT +brief course ADT]
2nd Rec. PSMA-PET:
Oligometastatic recurrence.
(ADT)
11 (69) Localized PrCa
GG3, PSA 5.2, cT2a
RT
[mHSPC]
25. Common iliac
LN
26. Aorto-caval LN
25. 25 Gy/5
26. 25 Gy/5
No 15.6
15.6
25. Yes (*)
26. Yes (*)
Baseline BS/CT:
[MDRT]
2nd Rec. PSMA-PET:
Poly-metastatic recurrence;
(ADT)
A. Mesci et al.

Clinical and Translational Radiation Oncology 39 (2023) 100583
4
RT-qPCR
For tumor RNA extraction, frozen 22RV1 xenograft tumors were
crashed and homogenized using a Precellys 24 tissue homogenizer
(Rockville, MD). Total RNA from homogenized tissues, or cells collected
from tissue cultures, was isolated using TRIzol (Life technologies, Grand
Island, NY) and RNeasy kit (QIAGEN) column were utilized for RNA
purication. cDNA was prepared and RT-qPCR was performed using a
MBI Corbett Rotor Gene 6000 (Dorval, QC), as described [22]. Each
sample was run in duplicate for a total of 45 cycles. Relative gene
expression was calculated using Livak comparative Ct (2
-ΔΔCt
) method
[23], where values were normalized to a housekeeping gene (s18).
TaqMan probes used: Catalog ID: 18 s: Hs03003631_g1 and FOLH1
Hs00379515_m (Life Technologies, CA).
Immunoblotting
PrCa lines seeded in 6-well plates (at 3-7x10
5
cells/well) were
treated with 0, 2 or 8 Gy in one fraction and incubated for 48 h. Cells
were washed, lysed and subjected to immunoblotting, using specic
primary antibodies against PSMA, neuron-specic enolase (NSE) or,
GAPDH (Cell Signaling, as described earlier) [21]. Antibodies were
purchased from Cell Signaling Technology (Whitby, ON). Immuno-
reactions were visualized with ECL (Bio-Rad, CA) and exposed to a
Vilber fusion-FX imaging system (Marne-la-Vall´
ee, France).
Immunohistochemistry (IHC)
FFPE tissue blocks were sectioned in 5
μ
M thickness slices, de-
parafnized and rehydrated in xylene and ethanol, followed by endog-
enous peroxidase removal, and heat antigen retrieval in citrate buffer.
Tissues were blocked in 10 % goat serum and incubated with non-
specic serum or anti-PSMA, anti-NSE, anti-phospho-histone H3 (P-
H3-Ser-10) and anti-a-Methylacyl-CoA Racemase (AMACR) antibodies,
Cell Signaling Technology (Whitby, ON). This was followed by incuba-
tion with biotinylated goat-anti-rabbit secondary antibody and strepta-
vidin peroxidase and developed using Nova Red (Vector Labs, CA).
Hematoxylin was used as counter stain.
Quantication of IHC markers: Marker expression was quantied in
IHC specimens with the H-score system, which is considered as one of
the “gold standard” methods in IHC quantication. H-Scores were
determined by multiplication of the percentage of cells with staining
intensity ordinal value (0 for no, 1 for weak, 2 for medium, 3 for strong),
ranging from 0 to 300 possible values, [H-score =(% weak staining) (1)
+(% medium staining) (2) +(% heavy staining) (3)]. Typically, ten
random high-power elds were assessed from whole xenograft sections,
4–6 tumors per treatment group.
Statistical analysis
Unpaired T-test, one- or two-way ANOVA with post hoc Tukey’s
multiple comparison tests were used for statistical analysis. Analysis was
pursued using GraphPad prism v9.5. Signicance was accepted at p ≤
0.05 (*=p <0.05, **=p <0.01, ***=p <0.001 and ****=p <0.0001).
Results
PSMA-PET – Based assessment of treatment response to metastasis-
directed RT (MDRT).
In a total of 11 patients, three of them presented with de novo met-
astatic disease and eight with localized PrCa. Four patients were initially
treated with radical prostatectomy and four with radical radiotherapy to
prostate. 26 courses of MDRT were given to a total of 25 metastatic le-
sions (one lesion was re-treated, see Table 1) followed by
18
F-DCFPyL
PET/CT (PSMA-PET) scan upon biochemical disease recurrence. All
patients were offered standard of care ADT with or without androgen
receptor-axis-targeted therapy (ARAT) at the time of MDRT, but 5 out of
11 patients declined. All patients were staged with CT of chest,
abdomen, and pelvis and bone scans prior to MDRT, while 4 were also
staged with PSMA-PET. Distribution of the lesions was: 1 regional lymph
node (in the presence of other, metastatic lesions), 6 non-regional lymph
nodal (M1a), 15 bone (M1b), 3 visceral (lung; M1c). One patient met the
denition of metastatic castrate-resistant PC (mCRPC) at the time of
PSMA-PET detected recurrence. A total of three patients were on com-
bined androgen deprivation therapy (ADT: LHRH-agonist) and androgen
receptor axis therapy (ARAT: abiraterone and prednisone or Enzaluta-
mide) at the time of baseline as well as during the repeated conventional
imaging or PSMA-PET, while the remaining eight patients were on no
systemic therapy at either time. RT doses ranged from 20 Gy in 5 frac-
tions (BED
1.5
=73) to 48 Gy in 4 fractions (BED
1.5
=432). No grade 3 or
higher toxicity was noted in any of the patients. Interestingly, PSMA-
PET revealed no uptake after radiotherapy in 18 out of the 26 courses.
Fig. 1 illustrates representative images of metastatic sites imaged with
baseline PSMA-PET/CT before MDRT, RT dose distributions, and sec-
ondary PSMA-PET/CT at biochemical progression after MDRT. CT and
bone scans were concordant with improvement in all 18 lesions that
responded. All patients showed biochemical response (decreasing PSA)
in response to MDRT. However, eventually all cases experienced
biochemical failure and developed additional metastatic sites (5 oligo-
metastatic, 6 poly-metastatic). One patient had further loco-regional
failure. All patients were offered systemic therapy while 7 received
further MDRT.
PSMA expression patterns in human PrCa
We questioned whether the observed “response” could be an artifact
of altered PSMA-PET due to reduced PSMA expression in irradiated
metastatic PrCa. Given the challenges involved in obtaining biopsies
from metastases, we investigated effects of RT on PSMA expression in
preclinical PrCa models. However, rst, we assessed PSMA expression in
a number of radical prostatectomy and diagnostic prostate biopsy
specimens (obtained with standard transrectal ultrasound (TRUS)-
guidance) to compare to preclinical models. Fig. 2 shows representative
images of H&E, PSMA and
α
-methylacyl-CoA racemase (AMACR) IHC
staining of prostatectomy and core needle biopsy tissue (ISUP grade
group 2 adenocarcinoma, Gleason patterns 3 and 4), revealing hetero-
geneous PSMA expression, ranging from negative to strongly positive in
malignant glands (high power 40x magnication). Malignant glands
were conrmed by AMACR staining, commonly used for conrmation of
carcinoma [24] (See Fig. s1 for low magnication images). PSMA
expression was heterogeneous in the human tissue, with some glands
expressing high levels (red arrows) and others no (negative, blue
RP: radical prostatectomy; RT: radiotherapy; ADT: androgen deprivation therapy; ARAT: androgen receptor axis therapies; 2nd: secondary; 3rd: tertiary; Rec.:
recurrence; m: metastatic; NH: hormone naïve; HSPC: hormone sensitive prostate cancer; CRPC: castrate-resistant prostate cancer.
*No uptake above background.
**
MDRT given after 2nd PSMA scan.
$
Metastatic lesion response detected with PSMA-PET at time of secondary (2nd) or tertiary (3rd) biochemical recurrence, one lesion showed initially response (2nd
Rec. PSMA-PET) but avidity at tertiary progression (3rd Rec. PSMA-PET).
&: indicates cases that were on systemic therapy (ADT +ARAT) at the time of baseline and repeated conventional imaging or PSMA-PET.
A. Mesci et al.

Clinical and Translational Radiation Oncology 39 (2023) 100583
5
arrows) PSMA, conrming a signicant intra-tumor heterogeneity.
Early and late effects of low BED radiotherapy on PSMA protein and
mRNA levels
To study effects of radiation treatment on PSMA expression, we
compared baseline expression of PSMA in commonly used castration-
sensitive (LNCap), castrate-resistant (22RV1) and neuroendocrine
castrate-resistant (PC3 and DU145) PrCa cell lines. Concordant with
previous reports [25], immunoblotting showed that LNCap and 22RV1
cells expressed signicant levels of PSMA protein but no expression in
neuroendocrine PC3 and DU145 cells (Fig. 3A-B). To assess the inu-
ence of RT, cells were irradiated with 2 Gy or 8 Gy and analyzed 48 h
later. In LNCap cells, 8 Gy RT resulted in a small but signicant decrease
of PSMA protein levels (0.88 ±0.14-fold and 0.67 ±0.06-fold for 2 Gy
and 8 Gy, respectively). GAPDH levels were not altered in response to
RT. Conversely, there was a non-signicant trend for increase in PSMA
levels of 22RV1 (1.28 ±0.21 and -fold 1.16 ±0.29-fold, for 2 and 8 Gy,
respectively). Similarly, we detected non-signicant trends for change in
PSMA mRNA levels after RT (LNCAp: 0.92 ±0.11-fold and 22RV1: 1.55
±0.26-fold; Fig. 3C) and no expression in DU145 or PC3 cells after RT
(Fig. 3A).
Since transformation of PrCa into neuroendocrine phenotype is
associated with decreased PSMA levels [12], we examined in PrCa cells
the levels of neuron-specic enolase (NSE). Consistent with other re-
ports [26], LNCap cells expressed NSE weakly while 22RV1 cells showed
high levels of NSE expression (Fig. 3A-B). Castrate-resistant neuroen-
docrine DU145 and PC3 cells showed signicant levels of NSE, which
increased with RT in DU145 (4.07 ±0.88-fold for 2 Gy and 4.00 ±1.07-
fold expression for 8 Gy) and to a lesser degree, in PC3 cells (1.37 ±
0.49-fold for 2 Gy and 2.03 ±0.49-fold expression for 8 Gy) (Fig. 3A-B).
PSMA expression in xenografts of human PrCa
We generated xenografts of PC3 and 22RV1 cells in parallel and
treated them with 0 Gy (mock) or 5 Gy of radiation treatment (schematic
Fig. 4A). PSMA expression was readily detectable in 22RV1 but not PC3
xenografts (Fig. 4B-C). To appreciate the effects of RT treatment on
PSMA expression, we quantied H-scores for each of the stains.
Concordant with in vitro ndings, RT (5 Gy) did not alter signicantly
PSMA protein levels in xenografts (Fig. 4D; H-scores: 22RV1: Control:
25.0 ±14.4 vs RT: 25.7 ±3.9; PC3: Control:1.0 ±1.0 vs RT: 0.0 ±0.0).
Both 22RV1 and PC3 xenografts expressed NSE, with no spatial corre-
lation between PSMA and NSE expression in the 22RV1 cells. To test if
PSMA expression is associated with regions of tumor proliferating
rapidly, we examined the distribution of phosphorylated histone-H3 (P-
H3), an established marker of DNA replication and mitosis [27]
(Fig. 4E). While PSMA staining showed heterogeneous expression, there
was no detectable association of PSMA expression with the distribution
of mitotically active foci (P-H3 stain). RT (5 Gy) did not alter PSMA
expression nor its association with P-H3. Thus, in PrCa xenograft tumors
we detected no evidence of acute regulation of PSMA by single fraction
Fig. 1. Representative baseline PSMA-PET, metastasis-directed RT (MDRT) dose distributions, and secondary recurrence PSMA-PET images. Representative
images of patient #6 are shown illustrating baseline and 2nd recurrence (Rec.) PSMA-PET (post-MDRT) of left iliac bone metastasis (top row), left perirectal lymph
node metastasis (middle row) and hilar lymph node metastasis (bottom row). RT dose distribution is illustrated in the middle column.
A. Mesci et al.

Clinical and Translational Radiation Oncology 39 (2023) 100583
6
low BED RT and no clear correlation between the expression of neuro-
endocrine (NSE), mitosis (P-H3) markers and PSMA.
Long term effects of high BED RT on PSMA expression - models of radio-
resistance
Next, we examined whether acquired radio-resistance, developed
with survival after high BED RT, could modulate PSMA expression. For
that, we analyzed two PrCa cellular models of acquired radio-resistance
22RV1-RR and DU145-RR previously developed and characterized [20]
(see Methods). Fig. s2A illustrates the radio-resistant properties of
22RV1-RR cells to increasing RT doses compared to parental cells.
Xenograft studies show similar growth kinetics for 22RV1 and 22RV1-
RR (Fig. s2B). However, 22RV1-RR xenografts demonstrate resistance
Fig. 2. SMA expression in human prostate tumor specimens. PSMA expression in human prostate tumors is highly heterogeneous. (A.) Representative images of a
human prostatectomy specimen (40x magnication). The patient was diagnosed with ISUP Grade Group 2 adenocarcinoma, acinar type. Three representative tumor
areas are shown in three rows. Tumor sections were stained with Hematoxylin & Eosin (H&E; left column) or subjected to IHC for
α
-methylacyl-CoA racemase
(AMACR; middle column), and PSMA (right column). Red arrows indicate malignant glands with PSMA staining; blue arrows indicate malignant glands without
PSMA staining. (B.) Representative sections of human diagnostic prostate core biopsies. The patient was diagnosed with ISUP Grade Group 2 (Gleason score (GS) 3 +
4) adenocarcinoma. Red arrows indicate fused GS4 glands with high PSMA expression; blue arrows indicate fused glands lacking PSMA expression. (For interpre-
tation of the references to colour in this gure legend, the reader is referred to the web version of this article.)
A. Mesci et al.

Clinical and Translational Radiation Oncology 39 (2023) 100583
7
to acute radiation treatments (5 Gy) compared to parental, also reected
in the survival (time to tumor endpoint) of the host animal (Fig. s2C).
Immunoblotting and qPCR analysis of untreated 22RV1-RR cells
showed substantially reduced PSMA levels compared to parental
(22RV1: 0.17 ±0.09-fold protein and 0.62 ±0.07-fold transcript),
while PSMA levels in DU145-RR cells remained undetectable (Fig. 5A-
B). 22RV1-RR but not DU145-RR cells appeared to express lower levels
of NSE compared to wild-type counterparts (Fig. 5A-B). Similarly, qPCR
demonstrated 54 % lower PSMA (FOLH1) mRNA level in 22RV1-RR over
22RV1 (Fig. 5C). We conrmed these results with IHC and qPCR in
xenografts (Fig. 5D-F). Similar to tissue culture results, 22RV1-RR xe-
nografts expressed substantially lower levels of PSMA protein and
mRNA levels (Fig. 5D-F). Further, low BED RT (5 Gy) did not acutely
alter the expression levels of PSMA or NSE in 22RV1-RR tumors (Fig. 5D-
E).
LNCap
LNCap control
22RV1 control
LNCap RT (5Gy)
22RV1 RT (5Gy)
Fig. 3. PSMA expression in prostate cancer cell lines and its modulation by radiation treatment. (A.) Immunoblotting analysis of PSMA, neuron-specic
enolase (NSE) and GAPDH in untreated and irradiated (0, 2 or 8 Gy) LNCAp, 22RV1, DU145, and PC3 cells. Representative immunoblots of three independent
experiments are shown. (B.) Immunoblot densitometric analysis of the experiments in (A.) for PSMA and NSE. Densitometry values for PSMA and NSE in each sample
were normalized to GAPDH. Individual values were then normalized to the mean value of control (mock-radiated) samples. Graph shows Mean ±SE of 3 inde-
pendent experiments; * p <0.05, *** p <0.001; two-way ANOVA test). (C.) RT-qPCR analysis of PSMA (FOLH1 gene). ΔCT values were obtained by normalization to
18S ribosomal RNA. Graph shows Mean ±SE values for LNCAp and 22RV1 cells treated with 0 Gy (mock) or 5 Gy RT of 3 independent experiments (differences were
not statistically signicant).
A. Mesci et al.

Clinical and Translational Radiation Oncology 39 (2023) 100583
8
Discussion
Several reports have shown improved sensitivity and specicity of
PSMA-PET compared to conventional imaging at various stages of PrCa.
However, use of this modality in assessing response to MDRT is less
commonly reported. Baumann et al. [15] reported a small series of
patients assessed by
68
Ga-PSMA-11 after ablative dose MDRT. They
found a high response rate, similar to our report. In contrast, here we
report PSMA-PET response to both ablative and palliative doses of RT
(BED
1.5
=73.3–432 Gy). Regardless, our cohort also showed high rates
of local disease control in treated metastases. Importantly, lesions
showing residual PSMA avidity were not limited to those treated with
22RV1 control
22RV1 RT (5Gy)
PC3 control
PC3 RT (5Gy)
Fig. 4. PSMA expression in untreated and irradiated prostate cancer xenografts. (A.) Schematic diagram of xenograft treatments (Created using BioRender.
com). Prostate cancer cells were grafted ectopically in the right ank. RT treatment (1x 5 Gy) was delivered when xenografts reached approximately 100 mm
3
.
Animals were euthanized when tumours reached 2200 mm
3
, tumors were collected, bisected and were either formalin-xed parafn-embed (FFPE) or snap frozen for
IHC or RNA extraction, respectively. (B., C.) PSMA and NSE IHC analyses of untreated and irradiated parental 22RV1 (B.) and PC3 (C.) xenografts (imaged at 40x
magnication). (D.) H-score analysis of PSMA staining in untreated and irradiated xenografts. (E.) Representative images of PSMA and phospho-histone H3 (P-H3)
IHC analysis of mock-treated and irradiated (1x 5 Gy) 22RV1 xenografts (at 40x magnication). No association could be detected between PSMA and P-H3 stain with
analysis of whole xenograft section slides (10 random high-power elds, 4–6 xenografts per group were analyzed) (quantication not shown).
A. Mesci et al.

Clinical and Translational Radiation Oncology 39 (2023) 100583
9
conventional palliative RT doses. Further studies are required to deter-
mine whether high dose RT provides superior local control.
Baumann et al. [15] observed that a longer interval between RT and
PSMA may result in a greater reduction of PSMA avidity. Interestingly,
the interval between RT and PSMA-PET was generally longer in our
study but the number of lesions showing residual PSMA signal was
comparable (4/18 vs 8/26). The optimal timing of PSMA-PET imaging
after RT is unclear and may inuence observed response rates. It is
possible that
18
F-DCFPyL, used in this study, may offer improved
sensitivity and signal-to-noise ratio over
68
Ga-based tracers [15,28].
However, we feel it is unlikely that the choice of specic tracer would
have signicantly changed the observations in these studies. Further,
use of systemic therapy (ADT and/or ARATs) for the management of
metastatic PrCa, before re-staging, may indeed inuence the ability of
PSMA-PET to detect residual surviving disease at metastatic sites treated
with MDRT. Nevertheless, this factor did not contribute to the obser-
vations in our series since none of the patients received new systemic
treatments at the time of evaluation with PSMA-PET.
To date, access to PSMA-PET imaging remains limited in many
countries and PSMA-based imaging has not been routinely incorporated
in clinical trial protocols. Therefore, similar to our series, the majority of
patients with available PSMA-PET imaging would have scans mainly at
the time of biochemical recurrence but not baseline PET. There is an
increasing need for standardization of PSMA-PET reporting. Evaluation
of response to RT presents an evolving challenge and reporting of sites
with partial response would be more challenging. For that, in this report
we did not provide rigid terms to describe the observed signals but give
SUV values when residual signal is detected. Larger studies are needed to
help standardize reporting for such patients. We hope that the ndings
of this study could help improve the biological prospective of reporting
specialists.
PSMA expression levels in tumors were shown to have consequences
for PSMA-PET detection [13] and understanding the mechanisms
regulating PSMA expression is important. Our analysis of PSMA
expression at the molecular level, in human PrCa xenografts, conrms a
similar heterogeneous pattern of expression as in human prostate tu-
mors. This heterogeneity suggests caution in considering PSMA as a
universal tumour marker and guide for MDRT given the absence of
PSMA expression from signicant portions of tumors (Fig. 2,4,5). Pre-
vious reports also observed signicant variation of PSMA expression
within tumors, and between primary prostate tumors and PrCa metas-
tases in the same patient [14,29]. Tsourlakis et al. [30] found that PSMA
expression was present at least weakly in 1144/1172 tissue spots
derived from 173 prostatectomy patients, but noted signicant
Fig. 4. (continued).
A. Mesci et al.

Clinical and Translational Radiation Oncology 39 (2023) 100583
10
expression variances (i.e. strong and weak staining present within the
same tumour) in just over 50 % of the analyzed tumour samples.
Technical factors (e.g. tissue processing, staining) may account for some
differences between reports. Further studies are crucial in understanding
the mechanisms regulating PSMA in tumors. Our attempts to correlate
markers of neuroendocrine differentiation (NSE) or DNA replication (P-
H3 histone) did not show correlation with PSMA expression (Fig. 4).
Despite widespread use of PSMA-PET after RT, the regulation of
PSMA expression by RT was not investigated until recently. As discussed
above, while this study was being prepared for publication, Sheehan
et al. [19] reported upregulation of PSMA protein, but not mRNA levels,
by fractionated RT in castrate-sensitive (LNCap) and castrate-resistant
(22RV1) cells and PDX tumour models. They postulated that PSMA
expression may be regulated by RT mostly at the post-transcriptional
level. That study investigated effects of low BED fractionated RT
(5–28 Gy,
α
/β ratio of 1.5) and analyzed cells and tumors 1–2 weeks
after RT. In the present study, we also found trends of PSMA protein
upregulation in 22RV1 cells 48 h after RT (BED: 4.6–50 Gy). However,
we observed down-regulation of PSMA protein levels in LNCap in
response to the same treatments, which reached statistical signicance.
Fig. 5. PSMA expression in radiation-resistant clones of prostate cancer cells and xenografts. (A.) Representative immunoblots of PSMA, neuron-specic
enolase (NSE) and GAPDH expression in parental 22RV1 (22RV1-P), radio-resistant 22RV1 (22RV1-RR), parental DU145 (DU145-P), radio-resistant of DU145
(DU145-RR). (B.) Densitometric analysis of PSMA and NSE immunoblots (Mean ±SE of 3 independent experiments; * p <0.05, **** p <0.0001; one-way ANOVA).
(C.) RT-qPCR analysis of PSMA gene (FOLH1) expression in 22RV1-P and 22RV1-RR cells. ΔCT values were obtained by normalization to 18S ribosomal RNA (Mean
±SE of 3 independent experiments; * p <0.05; unpaired T-test). (D.) Representative images of IHC analysis of 22RV1-P and 22RV1-RR xenografts (40x magni-
cation) (tumor growth curves are shown in Fig.s2, 6 animals per treatment group). PSMA and NSE staining are shown for mock- (0 Gy) or radiation-treated (1 ×5
Gy) tumors. (E.) H-score analysis of PSMA staining in mock-treated or irradiated xenografts above (Mean ±SE of 3 xenografts analyzed ×10 high power elds
quantied per xenograft). (F.) RT-qPCR analysis of PSMA (FOLH1 gene) expression in 22RV1-P and 22RV1-RR xenografts. ΔCT values were obtained by normal-
ization to 18S ribosomal RNA. (Mean ±SE, 6 xenograft tumors per group; * p <0.05, **** p <0.0001; unpaired T-test).
A. Mesci et al.

Clinical and Translational Radiation Oncology 39 (2023) 100583
11
The changes were associated with similar trends in mRNA levels that
were not statistically signicant (Fig. 3). Further, we observed no sig-
nicant modulation of PSMA protein levels in 22RV1 xenografts
analyzed 18–30 days after low BED RT and these treatments did not alter
the undetectable levels of PSMA expression in neuroendocrine PrCa cells
and xenografts (PC3 and DU145) (Figs. 3-4). Although the observations
in 22RV1 cells in culture were similar between the two studies, the
ndings of our study do not support a signicant early regulation of
PSMA levels in PrCa by low BED RT.
Importantly, here we show that PrCa cell clones that survived high
BED RT express substantially reduced PSMA protein levels. The loss of
PSMA expression was not only detected at the protein but also at the
22RV1 RT (5Gy)
22RV1 control
22RV1-RR control
22RV1-RR RT (5Gy)
Fig. 5. (continued).
A. Mesci et al.

Clinical and Translational Radiation Oncology 39 (2023) 100583
12
transcript level. This indicates that development of radio-resistance in
PrCa tumor cells involves a transcriptional reprogramming that could
include suppression of FOLH1 gene (Fig. 5). These ndings highlight a
signicant concern in utilizing PSMA-PET as a response assessment tool
after RT.
We feel that the results of this study are novel and of high clinical
interest. It cautions clinicians to interpret negative PSMA-PET scans
carefully in patients treated previously with RT. Consequently, our re-
sults and those by Baumann et al. [15] are subject to the same caveat;
lack of PSMA-PET signal alone cannot be reliably equated to lack of
surviving tumour. Numerous clinical trials have and continue to be
designed to take advantage of PSMA-PET in PrCa therapeutics. Since this
relies on tumor PSMA expression, a better understanding of the regu-
lation of PSMA expression, at the molecular level, by PrCa therapies as
well as driver mutations, is key to properly interpreting the results of
such trials.
Finally, our preclinical results, must also be interpreted with caution;
biology of PrCa cell lines can differ from that of human tumors. Ulti-
mately, this work supports further preclinical and clinical studies to
elucidate the mechanisms regulating PSMA expression that remain
poorly understood.
Conclusion
Consistent with conventional imaging and early biochemical control,
PSMA-PET illustrates high rates of local control of oligo-metastatic PrCa
after MDRT. However, clinicians should be cognizant of the high degree
of heterogeneity of PSMA expression in PrCa tumors that is not neces-
sarily associated with expression of neuroendocrine features. RT may
acutely alter PSMA expression in some PrCa models but in our hands this
modulation appears to be limited. Importantly, PrCa cell survival after
high dose RT could be associated with signicant loss of PSMA expres-
sion at both the protein and mRNA levels, raising concerns on the uti-
lization of PSMA-PET as a long-term effective response assessment tool
after MDRT. Future clinical protocol and translational study design
should aim to provide biospecimens and analysis methods that can
address reliably whether survival after curative RT indeed limits the
sensitivity of PSMA-PET to detect residual or recurrent PrCa in the
prostate or metastatic sites.
Funding
Supported by Hamilton Health Sciences Foundation grant.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ctro.2023.100583.
References
[1] McCarthy M, Francis R, Tang C, Watts J, Campbell A. A Multicenter prospective
clinical trial of (68)Gallium PSMA HBED-CC PET-CT restaging in biochemically
relapsed prostate carcinoma: oligometastatic rate and distribution compared with
standard imaging. Int J Radiat Oncol Biol Phys 2019;104(4):801–8.
[2] Hofman MS, Hicks RJ, Maurer T, Eiber M. Prostate-specic membrane antigen PET:
clinical utility in prostate cancer, normal patterns, pearls, and pitfalls.
Radiographics 2018;38(1):200–17.
[3] Hofman MS, Lawrentschuk N, Francis RJ, Tang C, Vela I, Thomas P, et al. Prostate-
specic membrane antigen PET-CT in patients with high-risk prostate cancer
before curative-intent surgery or radiotherapy (proPSMA): a prospective,
randomised, multicentre study. Lancet 2020;395(10231):1208–16.
[4] Pan KH, et al. Evaluation of 18F-DCFPyL PSMA PET/CT for prostate cancer: a
meta-analysis. Front Oncol 2020;10:597422.
[5] Szabo Z, Mena E, Rowe SP, Plyku D, Nidal R, Eisenberger MA, et al. Initial
evaluation of [(18)F]DCFPyL for prostate-specic membrane antigen (PSMA)-
targeted PET imaging of prostate cancer. Mol Imaging Biol 2015;17(4):565–74.
[6] Rousseau E, Wilson D, Lacroix-Poisson F, Krauze A, Chi K, Gleave M, et al.
A prospective study on 18F-DCFPyL PSMA PET/CT Imaging in biochemical
recurrence of prostate cancer. J Nucl Med 2019;60(11):1587–93.
[7] Silver DA, et al. Prostate-specic membrane antigen expression in normal and
malignant human tissues. Clin Cancer Res 1997;3(1):81–5.
[8] Neale JH, Bzdega T, Wroblewska B. N-Acetylaspartylglutamate: the most abundant
peptide neurotransmitter in the mammalian central nervous system. J Neurochem
2000;75(2):443–52.
[9] Silhavy J, et al. Dissecting the role of Folr1 and Folh1 genes in the pathogenesis of
metabolic syndrome in spontaneously hypertensive rats. Physiol Res 2018;67(4):
657–62.
[10] Israeli RS, et al. Expression of the prostate-specic membrane antigen. Cancer Res
1994;54(7):1807–11.
[11] Conteduca V, Oromendia C, Eng KW, Bareja R, Sigouros M, Molina A, et al. Clinical
features of neuroendocrine prostate cancer. Eur J Cancer 2019;121:7–18.
[12] Bakht MK, Derecichei I, Li Y, Ferraiuolo R-M, Dunning M, Oh SW, et al.
Neuroendocrine differentiation of prostate cancer leads to PSMA suppression.
Endocr Relat Cancer 2019;26(2):131–46.
[13] Ferraro DA, Rüschoff JH, Muehlematter UJ, Kranzbühler B, Müller J, Messerli M,
et al. Immunohistochemical PSMA expression patterns of primary prostate cancer
tissue are associated with the detection rate of biochemical recurrence with (68)
Ga-PSMA-11-PET. Theranostics 2020;10(14):6082–94.
[14] Mannweiler S, Amersdorfer P, Trajanoski S, Terrett JA, King D, Mehes G.
Heterogeneity of prostate-specic membrane antigen (PSMA) expression in
prostate carcinoma with distant metastasis. Pathol Oncol Res 2009;15(2):167–72.
[15] Baumann R, Koncz M, Luetzen U, Krause F, Dunst J. Oligometastases in prostate
cancer: metabolic response in follow-up PSMA-PET-CTs after hypofractionated
IGRT. Strahlenther Onkol 2018;194(4):318–24.
[16] Evans MJ, Smith-Jones PM, Wongvipat J, Navarro V, Kim S, Bander NH, et al.
Noninvasive measurement of androgen receptor signaling with a positron-emitting
radiopharmaceutical that targets prostate-specic membrane antigen. Proc Natl
Acad Sci USA 2011;108(23):9578–82.
[17] Mathy CS, Mayr T, Kürpig S, Meisenheimer M, Dolscheid-Pommerich RC, Stoffel-
Wagner B, et al. Antihormone treatment differentially regulates PSA secretion,
PSMA expression and 68Ga–PSMA uptake in LNCaP cells. J Cancer Res Clin Oncol
2021;147(6):1733–43.
[18] Meller B, Bremmer F, Sahlmann CO, Hijazi S, Bouter C, Trojan L, et al. Alterations
in androgen deprivation enhanced prostate-specic membrane antigen (PSMA)
expression in prostate cancer cells as a target for diagnostics and therapy. EJNMMI
Res 2015;5(1).
[19] Sheehan B, et al. Prostate-Specic membrane antigen expression and response to
DNA damaging agents in prostate cancer. Clin Cancer Res 2022;28(14):3104–15.
[20] Ghiam AF, Taeb S, Huang X, Huang V, Ray J, Scarcello S, et al. Long non-coding
RNA urothelial carcinoma associated 1 (UCA1) mediates radiation response in
prostate cancer. Oncotarget 2017;8(3):4668–89.
[21] Storozhuk Y, Hopmans SN, Sanli T, Barron C, Tsiani E, Cutz J-C, et al. Metformin
inhibits growth and enhances radiation response of non-small cell lung cancer
(NSCLC) through ATM and AMPK. Br J Cancer 2013;108(10):2021–32.
[22] Lally JSV, Ghoshal S, DePeralta DK, Moaven O, Wei L, Masia R, et al. Inhibition of
acetyl-CoA carboxylase by phosphorylation or the inhibitor ND-654 suppresses
lipogenesis and hepatocellular carcinoma. Cell Metab 2019;29(1):174–182.e5.
[23] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25(4):402–8.
[24] Jiang Z, Woda BA, Rock KL, Xu Y, Savas L, Khan A, et al. P504S: a new molecular
marker for the detection of prostate carcinoma. Am J Surg Pathol 2001;25(11):
1397–404.
[25] Laidler P, Duli´
nska J, Lekka M, Lekki J. Expression of prostate specic membrane
antigen in androgen-independent prostate cancer cell line PC-3. Arch Biochem
Biophys 2005;435(1):1–14.
[26] Marchiani S, Tamburrino L, Nesi G, Paglierani M, Gelmini S, Orlando C, et al.
Androgen-responsive and -unresponsive prostate cancer cell lines respond
differently to stimuli inducing neuroendocrine differentiation. Int J Androl 2010;
33(6):784–93.
[27] Juan G, Traganos F, James WM, Ray JM, Roberge M, Sauve DM, et al. Histone H3
phosphorylation and expression of cyclins A and B1 measured in individual cells
during their progression through G2 and mitosis. Cytometry 1998;32(2):71–7.
[28] Dietlein M, Kobe C, Kuhnert G, Stockter S, Fischer T, Schom¨
acker K, et al.
Comparison of [(18)F]DCFPyL and [(68)Ga]Ga-PSMA-HBED-CC for PSMA-PET
imaging in patients with relapsed prostate cancer. Mol Imaging Biol 2015;17(4):
575–84.
[29] Wright GL, Mayer Grob B, Haley C, Grossman K, Newhall K, Petrylak D, et al.
Upregulation of prostate-specic membrane antigen after androgen-deprivation
therapy. Urology 1996;48(2):326–34.
[30] Tsourlakis MC, Klein F, Kluth M, Quaas A, Graefen M, Haese A, et al. PSMA
expression is highly homogenous in primary prostate cancer. Appl
Immunohistochem Mol Morphol 2015;23(6):449–55.
A. Mesci et al.