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Irradiation of Glioblastoma Cells Increases CD147 Levels in their Extracellular Vesicles: Contribution to Increased MMP9 Activity in the Tumor Microenvironment
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Volume 99 Number 2S Supplement 2017 2012-2016 were retrospectively reviewed (nZ30). Patient age, sex, tumor histology, tumor size, tumor location, radiation fractionation regimen, use of concurrent chemotherapy and survival post-radiation were recorded. The lung tumor volumes on CBCT were measured using contouring software for treatment days spaced 7 days apart. The percent total tumor volume (TV) reduction was calculated from baseline to the last day of treatment using the formula: 100*(TV CBCTstart e TV CBCTend)/(TV CBCstart). The early percent TV reduction was calculated using the formula: 100*(TV CBCTstart e TV CBCTday7)/(TV CBCstart). Tumor BED was calculated using a/bZ10. Results: Of the 30 patients in the study, 70% had adenocarcinoma and 30% had squamous cell carcinoma histologies. Ten different fractionation schedules were used: 66 Gy/33 fx (nZ2), 66 Gy/30 fx (nZ5), 63 Gy/35fx (nZ3), 60 Gy/30 fx (nZ9), 60 Gy/24 fx (nZ1), 60 Gy/15 fxs (nZ1), 48.6 Gy/27 fxs (nZ1), 45 Gy/25 fx (nZ3), 45 Gy/15 fxs (nZ3), 40 Gy/15 fxs (nZ2). The median initial tumor volume was 50.47 cc (range: 3.82 to 350.75 cc). The mean total tumor volume reductions and mean early TV reduction per fractionation group (with n>3) were: 42.8% and 10.2% for 66 Gy/30 fxs (BEDZ85.8); 49.6% and 22.7% for 66 Gy/30 fxs (BEDZ80.5); 43.4% and 3.9% for 63 Gy/35 fxs (BEDZ74.34); 39.1% and 10.3% for 60 Gy/30 fxs (BEDZ72); 38.4% and 12.3% for 45 Gy/15 fxs (BEDZ58.5); and 51.9% and 13.7% for 45 Gy/25 fxs (BEDZ53.1). There was no correlation between BED and total or early TV reduction (rZ-0.12, pZ0.8 and rZ0.03, pZ0.9). Conclusion: Lung tumor volume reduction rates both early and at the end of treatment can be tracked using CBCT. The mean tumor volume reductions and mean early TV reduction vary among different fractionation groups. In the cohort of 30 patients, no correlation was detected between BED and total or early TV reduction (rZ-0.12, pZ0.8 and rZ0.03, pZ0.9). Ongoing are analyses on additional patients to detect whether a statistically significant difference can be detected between tumor volume reduction rates per fractionation scheme. Author Disclosure: S. Choi: None. M. Wahl: None. S.E. Braunstein: None.
3392 Irradiation of Glioblastoma Cells Increases CD147 Levels in their Extracellular Vesicles: Contribution to Increased MMP9 Activity in the Tumor Microenvironment N. Colangelo, S. de Toledo, and E. Azzam; Rutgers University New Jersey Medical School, Newark, NJ Purpose/Objective(s): Glioblastoma is a locally invasive primary cancer of the central nervous system. Current treatment modalities are unable to adequately limit this invasiveness, leading to a 95% recurrence rate. Moreover, although radiotherapy has produced the largest improvement in survival for these patients, it is also associated with increased tumor invasiveness, perhaps contributing to the high recurrence rate. This study found that CD147, a protein which is commonly overexpressed in glioblastoma, may contribute to this invasion, in particular the increased invasion after radiation exposure. Interestingly, CD147 works to promote invasion in cancer cells via intercellular communication: tumors secrete CD147 in extracellular vesicles (EVs) to induce neighboring cells to produce matrix metalloproteinases (MMPs), which can degrade the extracellular matrix. Materials/Methods: We irradiated T98G human glioblastoma cells with 8Gy of 137Cs g-rays, and collected EVs from the medium at 24h using serial ultracentrifugation. The EVs were characterized by cryo-electron microscopy, NanoSight, and immunoblots. They were either collected for biochemical analyses or added at a 3x concentration to SVG human astrocytes maintained in culture for 24 h. One-way ANOVA with p<0.05 was considered significant, and the Holm test was used to control for multiple comparisons. Results: Mass spectrometry analysis had CD147 in the top 1% of increased proteins in EVs from irradiated glioblastoma cells (IR-EVs) relative to EVs from control, non-irradiated glioblastoma cells (C-EVs).
This increase was validated by immunoblot. Notably, EVs were enriched in the active (highly glycosylated) form of the protein. In contrast, CD147 protein levels in the glioblastoma cells were not increased, and PCR demonstrated the mRNA levels had not changed e suggesting irradiated cells mobilize existing CD147 into EVs. We then examined the effects of glioblastoma cells on the microenvironment, reporting fold change in astrocytes receiving glioblastoma cell EVs relative to those receiving no EVs. All results have an nZ6 with p<0.01. Astrocytes receiving C-EVs or IR-EVs had 3-fold higher MMP2 activity as determined by zymography. The activity of MMP9 secreted by astrocytes increased 10-fold in response to receiving C-EVs, but increased 14-fold in response to IR-EVs e a significant difference. Knockdown of CD147 by shRNA in the glioblastoma cells rendered its levels in the EVs undetectable by immunoblot. The C-EVs from these CD147-knockdown glioblastoma cells could still produce a 3-fold increase in MMP2 and an 8-fold increase in MMP9 activity, but IR-EVs could no longer produce an increase in MMP9 activity, i.e. MMP9 activity was reduced from 14-fold to 7-fold of the control astrocytes’. Conclusion: Glioblastoma cells can increase CD147 levels in their EVs in response to irradiation, contributing to increased MMP9 activity, but not necessarily MMP2 activity, through intercellular communication with astrocytes. Author Disclosure: N. Colangelo: None. S. de Toledo: None. E. Azzam: None.
3393 Monitoring Circulating Tumor Cells during Chemoradiation Therapy for Locally Advanced Pancreatic Cancer K.C. Cuneo,1 Y. Wang,2 M.A. Morgan,1 L. Rivera,2 I. Lohse,1 T.S. Lawrence,1 and S. Nagrath2; 1University of Michigan, Department of Radiation Oncology, Ann Arbor, MI, 2University of Michigan, College of Engineering, Ann Arbor, MI Purpose/Objective(s): Circulating tumor cells (CTCs) can be isolated and quantified from the peripheral blood of patients. We hypothesized that early changes in the number of circulating tumor cells during a course of chemoradiation for pancreatic cancer are predictive of treatment response. Materials/Methods: We obtained blood samples from 15 patients at serial time points throughout a treatment course for locally advanced pancreatic cancer. All patients were concurrently enrolled on a phase I therapeutic trial. Patients on the study received threes cycles of gemcitabine (1000 mg/ m2 on day 1 and day 8 of each 21 day cycle). Cycles 2 and 3 were administered with concurrent radiation (52.5 Gy in 25 fractions) to the primary tumor. Blood samples were analyzed prior to any therapy (T1), after cycle 1 of chemotherapy alone (T2), after three weeks of chemoradiation (T3), and at follow up (T4). CTCs were captured using a nanomaterial-based microfluidic platform, the GO chip, which captures live CTCs with anti-EPCAM antibodies bound to graphene oxide nano-posts. Captured cells are stained directly on the chip with anti-cytokeratin (CK) antibodies and the positive cells are counted. We compared changes in the number of CTCs between time points using a paired Student’s t-test and calculated progression free survival (PFS) using the Kaplan Meier method. This study was performed under an institutional review board approved protocol and all patients signed informed consent prior to participation. Results: CTCs were successfully quantified from 14 of the 15 patients at T1, T2, and T3. Median number of CTCs captured per ml whole blood at each time point were: 28.5 for T1 (range 4-65), 22 for T2 (range 2-45), 23 for T3 (range 3-68), and 22.5 for T4 (range 5-65). There was a trend for a decrease in the number of CTCs during induction chemotherapy (T1 to T2, paired Student’s t-test p value Z 0.078). Nine patients had a decrease in CTC counts during cycle 1 of chemotherapy and 5 patients had an increase or no change in CTC counts during this interval (T1 to T2). Median PFS for patients who had a decrease in the number of CTCs during cycle 1 was 11.7 months compared to 6.1 months for patients who did not have a decrease (Wilcoxon p value Z 0.018, log rank p value Z 0.079). Seven patients had a decrease in CTCs counts during chemoradiation therapy and
Volume 99 Number 2S Supplement 2017 2012-2016 were retrospectively reviewed (nZ30). Patient age, sex, tumor histology, tumor size, tumor location, radiation fractionation regimen, use of concurrent chemotherapy and survival post-radiation were recorded. The lung tumor volumes on CBCT were measured using contouring software for treatment days spaced 7 days apart. The percent total tumor volume (TV) reduction was calculated from baseline to the last day of treatment using the formula: 100*(TV CBCTstart e TV CBCTend)/(TV CBCstart). The early percent TV reduction was calculated using the formula: 100*(TV CBCTstart e TV CBCTday7)/(TV CBCstart). Tumor BED was calculated using a/bZ10. Results: Of the 30 patients in the study, 70% had adenocarcinoma and 30% had squamous cell carcinoma histologies. Ten different fractionation schedules were used: 66 Gy/33 fx (nZ2), 66 Gy/30 fx (nZ5), 63 Gy/35fx (nZ3), 60 Gy/30 fx (nZ9), 60 Gy/24 fx (nZ1), 60 Gy/15 fxs (nZ1), 48.6 Gy/27 fxs (nZ1), 45 Gy/25 fx (nZ3), 45 Gy/15 fxs (nZ3), 40 Gy/15 fxs (nZ2). The median initial tumor volume was 50.47 cc (range: 3.82 to 350.75 cc). The mean total tumor volume reductions and mean early TV reduction per fractionation group (with n>3) were: 42.8% and 10.2% for 66 Gy/30 fxs (BEDZ85.8); 49.6% and 22.7% for 66 Gy/30 fxs (BEDZ80.5); 43.4% and 3.9% for 63 Gy/35 fxs (BEDZ74.34); 39.1% and 10.3% for 60 Gy/30 fxs (BEDZ72); 38.4% and 12.3% for 45 Gy/15 fxs (BEDZ58.5); and 51.9% and 13.7% for 45 Gy/25 fxs (BEDZ53.1). There was no correlation between BED and total or early TV reduction (rZ-0.12, pZ0.8 and rZ0.03, pZ0.9). Conclusion: Lung tumor volume reduction rates both early and at the end of treatment can be tracked using CBCT. The mean tumor volume reductions and mean early TV reduction vary among different fractionation groups. In the cohort of 30 patients, no correlation was detected between BED and total or early TV reduction (rZ-0.12, pZ0.8 and rZ0.03, pZ0.9). Ongoing are analyses on additional patients to detect whether a statistically significant difference can be detected between tumor volume reduction rates per fractionation scheme. Author Disclosure: S. Choi: None. M. Wahl: None. S.E. Braunstein: None.
3392 Irradiation of Glioblastoma Cells Increases CD147 Levels in their Extracellular Vesicles: Contribution to Increased MMP9 Activity in the Tumor Microenvironment N. Colangelo, S. de Toledo, and E. Azzam; Rutgers University New Jersey Medical School, Newark, NJ Purpose/Objective(s): Glioblastoma is a locally invasive primary cancer of the central nervous system. Current treatment modalities are unable to adequately limit this invasiveness, leading to a 95% recurrence rate. Moreover, although radiotherapy has produced the largest improvement in survival for these patients, it is also associated with increased tumor invasiveness, perhaps contributing to the high recurrence rate. This study found that CD147, a protein which is commonly overexpressed in glioblastoma, may contribute to this invasion, in particular the increased invasion after radiation exposure. Interestingly, CD147 works to promote invasion in cancer cells via intercellular communication: tumors secrete CD147 in extracellular vesicles (EVs) to induce neighboring cells to produce matrix metalloproteinases (MMPs), which can degrade the extracellular matrix. Materials/Methods: We irradiated T98G human glioblastoma cells with 8Gy of 137Cs g-rays, and collected EVs from the medium at 24h using serial ultracentrifugation. The EVs were characterized by cryo-electron microscopy, NanoSight, and immunoblots. They were either collected for biochemical analyses or added at a 3x concentration to SVG human astrocytes maintained in culture for 24 h. One-way ANOVA with p<0.05 was considered significant, and the Holm test was used to control for multiple comparisons. Results: Mass spectrometry analysis had CD147 in the top 1% of increased proteins in EVs from irradiated glioblastoma cells (IR-EVs) relative to EVs from control, non-irradiated glioblastoma cells (C-EVs).
This increase was validated by immunoblot. Notably, EVs were enriched in the active (highly glycosylated) form of the protein. In contrast, CD147 protein levels in the glioblastoma cells were not increased, and PCR demonstrated the mRNA levels had not changed e suggesting irradiated cells mobilize existing CD147 into EVs. We then examined the effects of glioblastoma cells on the microenvironment, reporting fold change in astrocytes receiving glioblastoma cell EVs relative to those receiving no EVs. All results have an nZ6 with p<0.01. Astrocytes receiving C-EVs or IR-EVs had 3-fold higher MMP2 activity as determined by zymography. The activity of MMP9 secreted by astrocytes increased 10-fold in response to receiving C-EVs, but increased 14-fold in response to IR-EVs e a significant difference. Knockdown of CD147 by shRNA in the glioblastoma cells rendered its levels in the EVs undetectable by immunoblot. The C-EVs from these CD147-knockdown glioblastoma cells could still produce a 3-fold increase in MMP2 and an 8-fold increase in MMP9 activity, but IR-EVs could no longer produce an increase in MMP9 activity, i.e. MMP9 activity was reduced from 14-fold to 7-fold of the control astrocytes’. Conclusion: Glioblastoma cells can increase CD147 levels in their EVs in response to irradiation, contributing to increased MMP9 activity, but not necessarily MMP2 activity, through intercellular communication with astrocytes. Author Disclosure: N. Colangelo: None. S. de Toledo: None. E. Azzam: None.
3393 Monitoring Circulating Tumor Cells during Chemoradiation Therapy for Locally Advanced Pancreatic Cancer K.C. Cuneo,1 Y. Wang,2 M.A. Morgan,1 L. Rivera,2 I. Lohse,1 T.S. Lawrence,1 and S. Nagrath2; 1University of Michigan, Department of Radiation Oncology, Ann Arbor, MI, 2University of Michigan, College of Engineering, Ann Arbor, MI Purpose/Objective(s): Circulating tumor cells (CTCs) can be isolated and quantified from the peripheral blood of patients. We hypothesized that early changes in the number of circulating tumor cells during a course of chemoradiation for pancreatic cancer are predictive of treatment response. Materials/Methods: We obtained blood samples from 15 patients at serial time points throughout a treatment course for locally advanced pancreatic cancer. All patients were concurrently enrolled on a phase I therapeutic trial. Patients on the study received threes cycles of gemcitabine (1000 mg/ m2 on day 1 and day 8 of each 21 day cycle). Cycles 2 and 3 were administered with concurrent radiation (52.5 Gy in 25 fractions) to the primary tumor. Blood samples were analyzed prior to any therapy (T1), after cycle 1 of chemotherapy alone (T2), after three weeks of chemoradiation (T3), and at follow up (T4). CTCs were captured using a nanomaterial-based microfluidic platform, the GO chip, which captures live CTCs with anti-EPCAM antibodies bound to graphene oxide nano-posts. Captured cells are stained directly on the chip with anti-cytokeratin (CK) antibodies and the positive cells are counted. We compared changes in the number of CTCs between time points using a paired Student’s t-test and calculated progression free survival (PFS) using the Kaplan Meier method. This study was performed under an institutional review board approved protocol and all patients signed informed consent prior to participation. Results: CTCs were successfully quantified from 14 of the 15 patients at T1, T2, and T3. Median number of CTCs captured per ml whole blood at each time point were: 28.5 for T1 (range 4-65), 22 for T2 (range 2-45), 23 for T3 (range 3-68), and 22.5 for T4 (range 5-65). There was a trend for a decrease in the number of CTCs during induction chemotherapy (T1 to T2, paired Student’s t-test p value Z 0.078). Nine patients had a decrease in CTC counts during cycle 1 of chemotherapy and 5 patients had an increase or no change in CTC counts during this interval (T1 to T2). Median PFS for patients who had a decrease in the number of CTCs during cycle 1 was 11.7 months compared to 6.1 months for patients who did not have a decrease (Wilcoxon p value Z 0.018, log rank p value Z 0.079). Seven patients had a decrease in CTCs counts during chemoradiation therapy and