Main Session
Sep
28
PQA 03 - Digital Health Innovation and Informatics, Patient Safety & Quality, and Radiation and Cancer Biology
2537 - Targeting Radiation-Triggered Cell-Surface GRP78 via Antibody-Drug Conjugate for Gastric Cancer
Presenter(s)
P.M. Quan Mai, BS - University of Texas Health Science Center at Houston, Houston, TX
P. M. Q. Mai1, P. C. Mallepadi2, P. Biswal1, N. T. Tra1, S. K. Samala3, B. M. Lakshmisha1, S. F. Ehsan4, A. Vasan1, L. W. Cheung2, G. Raja1, and S. Krishnan1; 1The University of Texas Health Science Center at Houston, Houston, TX, 2University of Texas Health Science Center at Houston, Houston, TX, 3All India Institute of Medical Sciences, New Delhi, India, 4UT MD Anderson UTHealth Houston, Houston, TX
Purpose/Objective(s):
Radiation can trigger tumor-specific cell-surface localization of proteins that can be leveraged in antibody-drug conjugate (ADC) therapies while sparing normal tissues. One such protein is 78-kDa Glucose-Regulated Protein (GRP78), which canonically resides in the endoplasmic reticulum but relocates to the cell surface upon radiation. In this study, we investigated the functional role of cell-surface GRP78 (csGRP78), elucidated the mechanism underlying the tumor-specific localization, and evaluated the therapeutic efficacy of an anti-GRP78 ADC in gastric cancer (GC) cells.Materials/Methods:
Irradiated human (AGS and MKN45), murine (YTN16) GC and normal (HUVEC and GES1) cells were subjected to flow cytometry and immunofluorescence for csGRP78 levels. Cancer phenotypes of csGRP78-positive (csGRP78+) cells was examined by cell growth assay and flow cytometry co-staining the cells with GRP78 and phenotypic markers. Mechanistic investigations of GRP78 cell-surface presentation were performed using immunoprecipitation and inhibitor experiments. To assess the biodistribution of the anti-GRP78 antibody, we injected GC xenografts with antibody-Alexa Fluor 647 dye prior to fluorescence imaging. Lastly, Annexin V/PI staining was performed for the efficacy after radiotherapy (RT) and anti-GRP78 ADC co-treatment.Results:
We detected RT-induced elevation of csGRP78 in all GC cells, notably not in the normal cells. The csGRP78+ population was enriched for Ki67 (proliferation marker), CD44 (stemness marker), and N-cadherin (metastasis marker) compared to csGRP78-negative population. The proliferative advantage of csGRP78+ cells was confirmed functionally by cell growth assay. We found that RT-induced csGRP78 was O-glycosylated and that this O-glycosylation was cancer-specific. GRP78 bound to the O-glycosylation initiator N-acetylgalactosaminyltransferase 2 (GALNT2) only in cancer cells but not in normal cells. Inhibition of GALNT2 using gene knockdown or inhibitor attenuated RT-induced GRP78 cell-surface translocation. In irradiated cancer cells, inhibition of microvesicle formation, but not exosomes, trapped cell-surface GRP78 onto the cell-surface, suggesting that GRP78 is exocytosed via microvesicles. This presents a unique opportunity to combine microvesicle inhibitors with radiation to anchor GRP78 on the cell-surface for improved ADC-homing. The in vivo models showed a significant accumulation of anti-GRP78 antibody in radiated tumors, whereas the amounts of the antibody in non-irradiated tumors and healthy organs were minimal. Moreover, RT and anti-GRP78 ADC co-treatment significantly decreased cell survival compared to RT or ADC alone. Consistent with an absence of csGRP78 in normal cells, the ADC had no effect on the viability of HUVEC cells.Conclusion:
Radiation-induced cell-surface expression of GRP78 enables selective targeting of cancer cells using ADCs.