Background
Methods
Prostate Cancer patients and healthy donors
Patient | Clinical Stage (all N0) | Gleason Score | Age (years) | Serum PSA ADT4 (ng/ml) | Serum PSA ADT12 (ng/ml) | Serum PSA at 6 months (ng/ml) |
---|---|---|---|---|---|---|
1 | T2b | 7 (3+4) | 66 | 10.5 | 2.10 | 1.2 |
2 | T2b | 7 (3+4) | 62 | 134.0 | 0.20 | <0.01 |
3 | T2 | 8 (3+5) | 70 | 8.3 | 1.40 | <0.1 |
4† | n/d | 7 (3+4) | 65 | 83.2 | 83.40 | † |
5 | T2c | 7 (3+4) | 69 | 95.2 | 4.10 | <0.1 |
6 | T2 | 8 (4+4) | 70 | 10.8 | 0.10 | <0.1 |
7 | T3a | 7 (3+4) | 53 | 36.5 | 7.20 | 0.3 |
8 | T3b | 6 (3+3) | 61 | 14.1 | 0.80 | 0 |
9 | T2 | 7 (4+3) | 66 | 21.1 | 0.20 | <0.1 |
10 | T2 | 8 (4+4) | 71 | 28.1 | 1.3 | n/d |
Urine sample collection
Patient | Time Point | Dip-Stick Blood, Protein, Glucose, Ketones, pH | Specimen Volume (ml) | Total Exosomes Recovered (μg) | Exosome Concentration (ng/ml) | ||||
---|---|---|---|---|---|---|---|---|---|
1
| ADT4 | 1 | 1 | 0 | 0 | 7 | 90 | 72.9 | 810.0 |
ADT12 | 0 | 1 | 0 | 0 | 5 | 180 | 141.9 | 788.3 | |
RT20 | 0 | 0 | 0 | 0 | 7 | 180 | 19.6 | 109.3 | |
2
| ADT4 | 1 | 1 | 0 | 0 | - | 170 | 125.5 | 738.2 |
ADT12 | 1 | 2 | 0 | 0 | 7 | 180 | 2.61 | 14.5 | |
RT20 | 0 | 0 | 0 | 0 | 5 | 90 | 39.2 | 435.7 | |
3
| ADT4 | 4 | 2 | 4 | 0 | 5 | 180 | 72.9 | 405.3 |
ADT12 | 2 | 1 | 1 | 0 | 5 | 180 | 70.9 | 393.9 | |
RT20 | 0 | 1 | 1 | 0 | 5 | 60 | 8 | 133.3 | |
4†
| ADT4 | 0 | 1 | 0 | 0 | 5 | 95 | 25.4 | 268.0 |
ADT12 | 1 | 3 | 3 | 0 | 5 | 55 | 6.54 | 118.9 | |
RT20 | - | - | - | - | - | - | - | - | |
5
| ADT4 | 4 | 0 | 0 | 0 | 5 | 180 | 38.4 | 213.6 |
ADT12 | 1 | 2 | 0 | 0 | 7 | 90 | 27.1 | 301.2 | |
RT20 | 1 | 1 | 0 | 0 | 6 | 150 | 5.1 | 34.5 | |
6
| ADT4 | 0 | 0 | 0 | 0 | 6 | 180 | 19.4 | 108.1 |
ADT12 | 1 | 0 | 0 | 0 | 5 | 180 | 6.2 | 34.7 | |
RT20 | 1 | 1 | 0 | 0 | 5 | 120 | 9.1 | 76.1 | |
7
| ADT4 | 3 | 1 | 1 | 0 | 6 | 97 | 39 | 402.1 |
ADT12 | 0 | 1 | 0 | 0 | 5 | 120 | 12.1 | 101.0 | |
RT20 | 1 | 1 | 0 | 0 | 5 | 45 | 17.7 | 395.1 | |
8
| ADT4 | 0 | 1 | 1 | 0 | 6 | 150 | 125.1 | 834.4 |
ADT12 | 0 | 1 | 0 | 0 | 5 | 110 | 26 | 236.4 | |
RT20 | 1 | 3 | 0 | 0 | 7 | 60 | 34.4 | 574.0 | |
9
| ADT4 | 0 | 1 | 0 | 0 | 5 | 120 | 8.2 | 68.3 |
ADT12 | 0 | 1 | 0 | 0 | 6 | 180 | 17 | 94.4 | |
RT20 | 2 | 3 | 4 | 0 | 6 | 60 | 133.1 | 2218.7 | |
10
| ADT4 | 0 | 1 | 0 | 0 | 5 | 120 | 19.4 | 162.3 |
ADT12 | 0 | 0 | 0 | 0 | 7 | 180 | 11.4 | 63.4 | |
RT20 | 0 | 0 | 0 | 0 | 6 | 170 | 88.3 | 519.4 |
Exosome purification
Cell culture
Electrophoresis and Immuno-blotting
Examining exosome membrane integrity
Examining proteolytic damage of exosomes by urine
Results
Purification of urinary exosomes
Changes in urine-exosome quantity during PCa therapy
Healthy Donor | Age of donor | Dip-Stick Blood, Protein, Glucose, Ketones, pH | Specimen Volume (ml) | Exosomes Recovered (μg) | Exosome Concentration (ng/ml) | ||||
---|---|---|---|---|---|---|---|---|---|
1
|
29
| 0 | 0 | 0 | 0 | 7 | 180 | 9.8 | 54.4 |
2
|
37
| 0 | 1 | 0 | 0 | 7 | 180 | 115.2 | 640.0 |
3
|
37
| 0 | 1 | 0 | 0 | 7 | 180 | 32.3 | 179.4 |
4
|
63
| 2 | 0 | 0 | 0 | 5 | 180 | 55.4 | 307.8 |
5
|
61
| 0 | 1 | 0 | 0 | 7 | 180 | 154.7 | 859.4 |
6
|
50
| 0 | 1 | 0 | 0 | 7 | 180 | 8.7 | 48.3 |
7
|
49
| 0 | 0 | 0 | 0 | 6 | 150 | 61.2 | 408.0 |
8
|
55
| 0 | 1 | 0 | 0 | 6 | 180 | 37.2 | 206.7 |
9
|
56
| 0 | 0 | 4 | 0 | 7 | 145 | 28.5 | 196.6 |
10
|
57
| 0 | 1 | 0 | 0 | 8 | 170 | 130.3 | 766.5 |
Prostate Cancer cell lines produce typical exosomes, positive for prostate and cancer-associated antigens
The phenotype of healthy donor urinary exosomes
Phenotype of PCa-patient's urinary exosomes, and evaluating changes with treatment
Exosome Markers | Cancer Marker | Prostate Markers | |||||||
---|---|---|---|---|---|---|---|---|---|
Patient | Time | CD9 | GAPDH | TSG101 | 5T4 | PSMA | PSA | Summary | |
LNCap
| N/A |
++++
|
+++
|
+++
|
+++
|
++++
|
++++
| The Comparator "Standard" Sample | |
Good Quality
|
p8
| ADT4 |
+++
|
++
|
++
|
++
|
++
|
+++
| Consistent, High Quality Exosomes. |
ADT12 |
+++
|
++
|
++
|
++
|
++
|
+
| Prostate markers diminish with treatment. | ||
RT20 |
+++
|
++
|
++
|
+
|
-
|
-
| 5T4 still evident at RT20 | ||
p7
| ADT4 |
++
|
+
|
++
|
+
|
+
|
+
| Good quality exosomes, but inconsistent, (increasing with treatment). | |
ADT12 |
++
|
++
|
+++
|
++
|
+
|
+
| Prostate markers & 5T4 still evident at RT20 | ||
RT20 |
+++
|
+++
|
+++
|
++
|
++
|
++
| |||
Intermediate Quality
|
p1
| ADT4 |
+
|
-
|
-
|
+
|
-
|
+
| Inconsistent, (increasing with treatment) |
ADT12 |
++
|
-
|
-
|
+
|
-
|
-
| Prostate markers barely detected, no clear pattern. | ||
RT20 |
+++
|
+
|
++
|
+
|
+
|
-
| 5T4 still evident at RT20 | ||
p3
| ADT4 |
+
|
+
|
-
|
+
|
-
|
-
| Inconsistent, (increasing with treatment) | |
ADT12 |
+
|
-
|
++
|
+
|
-
|
-
| Prostate markers absent. | ||
RT20 |
+++
|
++
|
-
|
++
|
-
|
-
| Strong 5T4 at RT20 | ||
Poor
|
p9
| ADT4 |
+++
|
+
|
+
|
+
|
+
|
+
| Inconsistent, (decreasing with treatment) |
ADT12 |
++
|
+
|
-
|
-
|
-
|
-
| Prostate markers barely detected, no clear pattern. | ||
RT20 |
+
|
-
|
-
|
-
|
+
|
-
| No 5T4 at RT20 | ||
p5
| ADT4 |
-
|
-
|
-
|
-
|
-
|
-
| Poor quality at 2/3 time-points | |
ADT12 |
-
|
-
|
-
|
-
|
-
|
-
| Not Evaluable | ||
RT20 |
+++
|
-
|
++
|
-
|
-
|
-
| |||
Very Poor Quality
|
p10
| ADT4 |
+++
|
++
|
+++
|
+
|
+
|
+
| Poor quality at 2/3 time-points |
ADT12 |
-
|
-
|
-
|
-
|
-
|
-
| Not Evaluable | ||
RT20 |
+
|
-
|
-
|
-
|
-
|
-
| |||
p6
| ADT4 |
+
|
-
|
-
|
-
|
-
|
-
| Poor quality at 3/3 time-points | |
ADT12 |
++
|
-
|
-
|
-
|
-
|
-
| Not Evaluable | ||
RT20 |
-
|
-
|
-
|
-
|
-
|
-
|