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1.
Fig 6

Fig 6. From: Definition of an enhanced immune cell therapy in mice that can target stem-like lymphoma cells.

Combining dual biotherapy with chemotherapy. Mice (BALB/c) bearing subcutaneous A-20 tumors were treated IP with 100mg/kg cyclophosphamide (CTX) either 14d before (A) or 2d after (B) IV treatment with PBS, CIK, vvDD or vvDD-CIK as before (n=8/group), tumor volume determined by caliper measurement.

Christopher H Contag, et al. Cancer Res. ;70(23):9837-9845.
2.
Fig 5

Fig 5. From: Definition of an enhanced immune cell therapy in mice that can target stem-like lymphoma cells.

(A) FVB mice with subcutaneous 6780 tumors were treated IT with vvDD or vvDD-CIK. Mice were sacrificed at indicated times and (i) degranulation assays run as before to determine levels of CD4+ cells activated by exposure to vvDD infected cells (n=3/group)(p<0.05) or (ii) vvDD neutralizing antibody assays were run on mouse plasma. (B) Human CIK cells alone, or following vvDD infection (MOI of 1.0 for 24h) underwent intracellular cytokine stains for the indicated cytokines, and mean fluorescence intensities determined (grey = CIK cells alone; black = CIK cells infected with vvDD); (D) ELISAs were run for IP-10 (CXCL10) or I-TAC (CXCL11) on the media from (i) human CIK cells; (ii) CIK cells infected with vvDD; (iii) OCI-Ly8 lymphoma cells exposed to CIK cells; (iv) OCI-Ly8 cells exposed to CIK cells infected with vvDD; (v) OCI-Ly8 cells alone.

Christopher H Contag, et al. Cancer Res. ;70(23):9837-9845.
3.
Fig 3

Fig 3. From: Definition of an enhanced immune cell therapy in mice that can target stem-like lymphoma cells.

(A) Experiment repeated as in Fig 2 with a second tumor cell line (6814). Percentages of animals that displayed relapse by 90 days after treatment are shown (n=7/group). (B) The mean fluorescence intensity of 6780 and 6814 cells stained with Sca-1 antibody, along with isotype (solid). (C) Experiment repeated as in Fig 2 with 6780-luciferase cells, only implantation was into NOD SCID mice. Relapse of mice treated iv with PBS or vvDD infected into CIK cells (C/V) is shown (Day0 = time of treatment and removal of doxycycline from regressed tumors) (n=3/group). (B) Animals from Fig 2 that were treated with CIK-vvDD dual therapy and that displayed no relapse by 90 days (n=4) were rechallenged with 6780-luciferase cells (1×106 IP). Subsequent BLI signal is shown.

Christopher H Contag, et al. Cancer Res. ;70(23):9837-9845.
4.
Fig 4

Fig 4. From: Definition of an enhanced immune cell therapy in mice that can target stem-like lymphoma cells.

(A) Mice that rejected re-challenge with 6780-luciferase cells (Fig 3D) were sacrificed after 90 days. Splenocytes were exposed to 6780 cells and de-granulation determined by CD107a or CD154 surface staining relative to naïve controls (for CD8 and CD4 cells respectively)(p=0.029 and 0.011 for CD4 and CD8). (B) FVB mice implanted IP with 6780-luciferase cells were treated when large primary tumors had formed with iv injections of (i) PBS (black); (ii) CIK cells (green); (iii) vvDD (blue); (iv) vvDD infected into CIK cells (red line), and subsequent tumor growth followed by BLI. When tumor BLI signal reached 1×108 Ph/sec/mouse, tumors were regressed through repression of MYC expression. Once tumors had regressed to undetetectable levels for >7 days, the MYC repression was removed, and relapse followed by BLI (survival curves are shown, with mice sacrificed when tumor burden reached 1×108 Ph/sec/mouse)(n=6/group)(*p<0.05).

Christopher H Contag, et al. Cancer Res. ;70(23):9837-9845.
5.
Fig 1

Fig 1. Targeting of pre-malignant lymphomas with CIK cells. From: Definition of an enhanced immune cell therapy in mice that can target stem-like lymphoma cells.

(A) 6780, 6814 or 8946 lymphoma cell lines expressing luciferase were treated with doxycycline to inactivate the MYC oncogene for the times indicated on the x-axis. At each time, effector (CIK cell) to tumor target mixes of 50:1, 25:1, 10:1 and 5:1 and tumor cells only, respectively, were left for 12 hours before BLI was used to determine tumor cell survival. (B) 6780 and 6814 cells treated with doxycycline for 48h, were stained with MULT-1 antibody, and mean fluorescence intensity determined by flow cytometry (black – no doxycycline; grey - with doxycycline; solid – isotype). (C) Trafficking of CIK cells to lymphoma. 6780 cells were injected subcutaneously in FVB mice and then underwent (i) no treatment; (ii) sustained MYC inactivation; (iii) MYC inactivation until tumors were no longer palpable, followed by MYC reactivation. All animals were treated iv with CIK cells expressing luciferase (isolated from FVB-luciferase transgenic animals) and imaged 72h later. Bioluminescence signal from the region of the tumor was determined (n=5/group), mean and standard deviation are shown.

Christopher H Contag, et al. Cancer Res. ;70(23):9837-9845.
6.
Fig 2

Fig 2. Treatment of minimal residual disease. From: Definition of an enhanced immune cell therapy in mice that can target stem-like lymphoma cells.

(A) Experimental procedure; FVB mice received intraperitoneal injections of 6780 cells expressing luciferase with subsequent tumor formation followed by BLI. Once tumors reached 1×108 Ph/Sec, MYC was inactivated to induce tumor regression. Once disease had been undetectable for at least 7 days, animals were treated with a single iv injection of (i) PBS; (ii) CIK cells; (iii) vvDD; (iv) CIK and vvDD or (v) vvDD pre-infected into CIK cells. At the same time doxycycline treatement was stopped (n=8/group, except PBS group, n=5). Subsequent relapse was followed by BLI. (B) Plots of individual animals' BLI signal over time after treatment is shown. Horizontal bars represent lower limits of detection. (C) Average day to relapse (first detectable BLI signal above background) after 6780-luciferase minimal disease was treated with CIK cells delivered iv at day 0 or day 0 and day 14 after doxycycline removal. PBS group 5 of 5 relapsed; CIK(d0) 6 of 7 relapsed; CIK(d0 &d14) 6 of 7 relapsed (* p=0.047).

Christopher H Contag, et al. Cancer Res. ;70(23):9837-9845.

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