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. 2020 Mar;59(3):323-332.
doi: 10.1002/mc.23157. Epub 2020 Jan 14.

Exosomes secreted by prostate cancer cells under hypoxia promote matrix metalloproteinases activity at pre-metastatic niches

Gagan Deep  1   2   3 , Anil Jain  4 , Ashish Kumar  1 , Chapla Agarwal  4   5 , Susy Kim  1 , W Matthew Leevy  6 , Rajesh Agarwal  4   5
Affiliations

Exosomes secreted by prostate cancer cells under hypoxia promote matrix metalloproteinases activity at pre-metastatic niches

Gagan Deep et al. Mol Carcinog. 2020 Mar.

Abstract

Approximately, 30 000 men die from prostate cancer (PCa) every year in the United States, mainly due to the metastasis. Thus, the key events associated with PCa metastasis are under rigorous investigation, with recent studies showing that preparation of pre-metastatic niches (PMN) in distant organs is an important step. However, the molecular basis for PMN preparation is still unclear. Hypoxia in primary tumors promotes aggressiveness; however, its precise role in metastasis is not clear. We recently reported that exosomes secreted by PCa cells under hypoxia promote stemness and invasiveness in naïve PCa cells; however, whether these extracellular vesicles also influence PMN remains unknown. In the present study, we isolated exosomes from human PCa PC3 cells under normoxic (21% O2 , exosomes secreted under normoxic condition [ExoNormoxic ]) and hypoxic (1% O2 , exosomes secreted under hypoxic condition [ExoHypoxic ]) conditions, and characterized their effect (10 µg exosomes, intraperitoneal (IP) treatment every 48 hours for 4 weeks) on key biomarkers associated with PMN in nude mice. Whole animal fluorescence imaging showed that ExoHypoxic treatment promotes matrix metalloproteinases (MMPs) activity in several putative metastatic sites. Histological studies confirmed that ExoHypoxic treatment enhanced the level of MMP2, MMP9, and extracellular matrix proteins (fibronectin and collagen) as well as increased the number of CD11b+ cells at selective PMN sites. Furthermore, proteomic profiling of exosomes by liquid chromatography/mass spectrometry identified cargo proteins in ExoNormoxic and ExoHypoxic as well as distinct canonical pathways targeted by them. These results suggest that exosomes secreted by PCa cells under hypoxia plausibly remodel distant PMN, and thus, could be a potential target to control metastatic PCa.

Keywords: exosomes; hypoxia; matrix metalloproteinases; pre-metastatic niches; proteomics.

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Conflict of interest statement

CONFLICT OF INTERESTS

The authors declare that there are no conflict of interests.

Figures

FIGURE 1
FIGURE 1
Human PCa ExoHypoxic promotes MMPs activity in selective organs in male athymic nude mice. A, Male athymic nude mice were injected with PBS, ExoNormoxic or ExoHypoxic (10 μg each, IP) on alternate days for 4 weeks. At the end, mice were injected with MMPsense 750 dye and imaged for fluorescence and X-ray by In Vivo Multispectral FX instrument. Representative images are shown for various groups (PBS, ExoNormoxic and ExoHypoxic). Mice without MMPsense injection served as negative control (bottom panel). In each image, dye in an eppendorf tube served as a positive control. B, Forty eight hours after MMPsense 750 dye injection, mice were killed, various organs isolated and imaged for fluorescence by In Vivo Multispectral FX imaging instrument. Organs: 1. brain; 2. lymph nodes + salivary glands; 3. lung + heart; 4. liver; 5. spleen; 6. kidneys; 7. prostate + seminal vesicles + bladder; 8. bone. Organs from animal without MMPsense dye injection served as negative control (top, left panel), while dye in an eppendorf served as positive control (in top left and bottom right images). C, Mice were injected IP with PBS or ExoGlow labelled exosomes (ExoNormoxic and ExoHypoxic) and various organs were imaged 24 hours later. Organs: 1. Brain; 2. Lung; 3. Heart; 4. Liver; 5.Spleen; 6. Kidney; 7. Prostate. ExoHypoxic, exosomes secreted under hypoxic condition; ExoNormoxic, Exosomes secreted under normoxic condition; IP, intraperitoneal; MMP, matrix metalloproteinases; PBS, phosphate buffered saline; PCa, prostate cancer [Color figure can be viewed at wileyonlinelibrary.com]
FIGURE 2
FIGURE 2
Human PCa ExoHypoxic promotes MMP2 and MMP9 expression in selective organs in male athymic nude mice. A,B, Male athymic nude mice were injected with PBS, ExoNormoxic or ExoHypoxic (10 μg each, IP) on alternate days for 4 weeks. At the end, mice were killed and mentioned organs were collected, processed, and analyzed for MMP2 and MMP9 expression by IHC as described in methods. Immunoreactivity for MMP2 and MMP9 was scored as 0+ (no staining), 1+ (weak staining), 2+ (moderate staining), 3+ (strong staining), 4+ (very strong staining). Data shown in the bar diagram represent mean ± SEM of immunoreactivity scores for ten randomly selected fields from 4 to 5 samples for each group. Representative images are shown at ×400. *P ≤ .05. ExoHypoxic, exosomes secreted under hypoxic condition; ExoNormoxic, Exosomes secreted under normoxic condition; IHC, immunohistochemistry; IP, intraperitoneal; MMP, matrix metalloproteinases; PBS, phosphate buffered saline; PCa, prostate cancer [Color figure can be viewed at wileyonlinelibrary.com]
FIGURE 3
FIGURE 3
Human PCa ExoHypoxic promote matrix remodeling in selective organs in male athymic nude mice. A,B, Male athymic nude mice were injected with PBS, ExoNormoxic or ExoHypoxic (10 μg each, IP) on alternate days for 4 weeks. At the end, mice were killed and mentioned organs were collected, processed, and analyzed for fibronectin and collagen expression by IHC. Immunoreactivity for fibronectin and collagen was scored as 0+ (no staining), 1+ (weak staining), 2+ (moderate staining), 3+ (strong staining), 4+ (very strong staining). Data shown in the bar diagram represent mean ± SEM of immunoreactivity scores for ten randomly selected fields from 4 to 5 samples for each group. Representative images are shown at ×400. *P ≤ .05. ExoHypoxic, exosomes secreted under hypoxic condition; ExoNormoxic, Exosomes secreted under normoxic condition; IHC, immunohistochemistry; IP, intraperitoneal; MMP, matrix metalloproteinases; PBS, phosphate buffered saline; PCa, prostate cancer [Color figure can be viewed at wileyonlinelibrary.com]
FIGURE 4
FIGURE 4
Effect of PCa ExoHypoxic treatment on CD11b+ cells in male athymic nude mice. Male athymic nude mice were injected with PBS, ExoNormoxic or ExoHypoxic (10 μg each, IP) on alternate days for 4 weeks. At the end, mice were killed and mentioned organs were collected, processed, and analyzed for CD11b+ cells by IHC and presented as % positive cells. Data shown in the bar diagram represent mean ± SEM of immunoreactivity scores for ten randomly selected fields from 4 to 5 samples for each group. Representative images are shown at ×400. *P ≤ .05. ExoHypoxic, exosomes secreted under hypoxic condition; ExoNormoxic, Exosomes secreted under normoxic condition; IHC, immunohistochemistry; IP, intraperitoneal; MMP, matrix metalloproteinases; PBS, phosphate buffered saline; PCa, prostate cancer [Color figure can be viewed at wileyonlinelibrary.com]
FIGURE 5
FIGURE 5
Characterization of proteins loaded in ExoNormoxic and ExoHypoxic by mass spectrometry. Proteomic profiling of ExoNormoxic and ExoHypoxic was performed by LC/MS/MS and data analyzed by IPA software. A, Cellular localization of proteins loaded in ExoNormoxic and ExoHypoxic are presented in pi diagrams. B, Top five canonical pathways in ExoNormoxic and ExoHypoxic are shown. In each case, percentage overlap with the dataset (left panel) as well as –log (P value; right panel) are presented. ExoHypoxic, exosomes secreted under hypoxic condition; ExoNormoxic, Exosomes secreted under normoxic condition; IPA, ingenuity pathway analysis; LC/MS/MS, liquid chromatography/mass spectrometry [Color figure can be viewed at wileyonlinelibrary.com]
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