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Rapamycin Promotes Mouse 4T1 Tumor Metastasis that Can Be Reversed by a Dendritic Cell-Based Vaccine.

Lin TJ, Liang WM, Hsiao PW, M S P, Wei WC, Lin HT, Yin SY, Yang NS - PLoS ONE (2015)

Bottom Line: Suppression of tumor metastasis is a key strategy for successful cancer interventions.However, rapamycin also exhibits immunosuppressant effects and is hence used clinically as an organ transplantation drug.We hypothesized that the immunosuppressive activities of rapamycin might also negatively mediate host immunity, resulting in promotion of tumor metastasis.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Injury Prevention and Control, Taipei Medical University, Taipei, Taiwan, ROC; Department of Neurosurgery, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan, ROC; Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan, ROC; Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan, ROC; Taiwan International Graduate Program (TIGP), Molecular and Biological Agricultural Sciences Program, Academia Sinica, Taipei, Taiwan, ROC.

ABSTRACT
Suppression of tumor metastasis is a key strategy for successful cancer interventions. Previous studies indicated that rapamycin (sirolimus) may promote tumor regression activity or enhance immune response against tumor targets. However, rapamycin also exhibits immunosuppressant effects and is hence used clinically as an organ transplantation drug. We hypothesized that the immunosuppressive activities of rapamycin might also negatively mediate host immunity, resulting in promotion of tumor metastasis. In this study, the effects of rapamycin and phytochemical shikonin were investigated in vitro and in vivo in a 4T1 mouse mammary tumor model through quantitative assessment of immunogenic cell death (ICD), autophagy, tumor growth and metastasis. Tumor-bearing mice were immunized with test vaccines to monitor their effect on tumor metastasis. We found that intraperitoneal (ip) administration of rapamycin after a tumor-resection surgery drastically increased the metastatic activity of 4T1 tumors. Possible correlation of this finding to human cancers was suggested by epidemiological analysis of data from Taiwan's National Health Insurance Research Database (NHIRD). Since our previous studies showed that modified tumor cell lysate (TCL)-pulsed, dendritic cell (DC)-based cancer vaccines can effectively suppress metastasis in mouse tumor models, we assessed whether such vaccines may help offset this rapamycin-promoted metastasis. We observed that shikonin efficiently induced ICD of 4T1 cells in culture, and DC vaccines pulsed with shikonin-treated TCL (SK-TCL-DC) significantly suppressed rapamycin-enhanced metastasis and Treg cell expansion in test mice. In conclusion, rapamycin treatment in mice (and perhaps in humans) promotes metastasis and the effect may be offset by treatment with a DC-based cancer vaccine.

No MeSH data available.


Related in: MedlinePlus

Vaccination with SK-TCL-activated DCs effectively suppresses rapamycin-promoted metastasis of 4T1 cells after resection of the primary tumor.(A), Mice were injected subcutaneously with 4T1-Luc2 cells (5 × 105 cells/100 μl PBS/mouse) into the mammary fat pad at day 0. At 16 days post tumor cell implantation, orthotopic primary tumors were surgically resected. Test mice were administered with control (saline) or rapamycin by intraperitoneal injection, and subsequently with or without vaccination by SK-TCL-mDCs by intravenous injection at indicated time points. (B), Representative bioluminescent images of tumor-resected mice (n = 8) in each group shown at 4 weeks post tumor resection. The red signal represents the highest level on the colorimetric scale. Labelling with “D” in photograph denotes the mice that had died before 4 weeks post tumor resection. (C), Quantification of tumor metastasis by measuring luciferase activity in p/s/cm2/sr in mice revealed along the indicated time points (n = 8). (D), Survival of test mice after different treatments. Labelling symbol for the control mouse group was intentionally marked with relatively large open circles in order to allow a clear indication of data sets obtained from each experimental group. *, values of P < 0.05; **, P < 0.01, were obtained between the Rapa only and Rapa + SK-TCL + mDCs groups. Similar results were obtained from three independent experiments.
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pone.0138335.g004: Vaccination with SK-TCL-activated DCs effectively suppresses rapamycin-promoted metastasis of 4T1 cells after resection of the primary tumor.(A), Mice were injected subcutaneously with 4T1-Luc2 cells (5 × 105 cells/100 μl PBS/mouse) into the mammary fat pad at day 0. At 16 days post tumor cell implantation, orthotopic primary tumors were surgically resected. Test mice were administered with control (saline) or rapamycin by intraperitoneal injection, and subsequently with or without vaccination by SK-TCL-mDCs by intravenous injection at indicated time points. (B), Representative bioluminescent images of tumor-resected mice (n = 8) in each group shown at 4 weeks post tumor resection. The red signal represents the highest level on the colorimetric scale. Labelling with “D” in photograph denotes the mice that had died before 4 weeks post tumor resection. (C), Quantification of tumor metastasis by measuring luciferase activity in p/s/cm2/sr in mice revealed along the indicated time points (n = 8). (D), Survival of test mice after different treatments. Labelling symbol for the control mouse group was intentionally marked with relatively large open circles in order to allow a clear indication of data sets obtained from each experimental group. *, values of P < 0.05; **, P < 0.01, were obtained between the Rapa only and Rapa + SK-TCL + mDCs groups. Similar results were obtained from three independent experiments.

Mentions: The results of Fig 3 further support the findings of our previous study using B16 cells [11] by once again demonstrating that shikonin-treated tumor cell lysate can be effectively employed to pulse and enhance mature dendritic cells (SK-TCL-mDC, Fig 3B and 3C) for therapeutic vaccination against metastasis of 4T1 tumors. As seen in Fig 2, we showed that in vivo treatment of mice with rapamycin promotes 4T1 tumor metastasis in our test mouse system. Therefore, next we were interested in whether rapamycin-promoted 4T1 cell metastasis after resection of the original primary tumor, could be suppressed by treatment with the SK-TCL-mDC cancer vaccine (Fig 4). In this experiment, an “anti-metastasis protocol” was designed in which the test DC vaccine was delivered on the very next day of tumor resection (Fig 4A). Bioluminescent imaging analysis of mice treated with control (saline) and different vaccine adjuvant formulations (mDCs only, SK-TCL-mDC, rapamycin only, and rapamycin + SK-TCL-mDC) were scored and analyzed. Consistent with the result of Fig 2, rapamycin treatment significantly increase the level of 4T1 tumor metastasis, as compared with the vehicle control treatment (P < 0.05) (Fig 4C), In contrast, SK-TCL-mDC treatment drastically reduced the tumor incidence and the rapamycin-promoted tumor metastasis, as evidenced by the lowest activity for metastasized tumors (Fig 4B and 4C). Tumor metastasis was suppressed by SK-TCL-mDC vaccination to a level of 62.5% at 2 months post tumor resection, as compared with the control group (P < 0.01) (Fig 4C). The level of anti-metastatic effect for rapamycin + SK-TCL-mDC treatment was similar to, i.e., not statistically different from, that of SK-TCL-mDC treatment (P = 0.94, n.s) (Fig 4C). The mean survival period of test mice with lung metastasis was 5, 12, 4 and 10 weeks for the control, SK-TCL-mDC, rapamycin, and rapamycin + SK-TCL-mDC group, respectively (Fig 4D). Co-treatment of rapamycin and SK-TCL-mDC vaccination (Rapa + SK-TCL-mDCs) significantly increased the life span of mice as compared to the vaccine-untreated (i.e., rapamycin alone) mice (P < 0.001), or the control group mice (i.e., untreated with DC vaccine and untreated with rapamycin) (Fig 4D). Together, these results clearly demonstrate that SK-TCL-mDC can effectively inhibit the initiation and progression of highly metastatic 4T1 malignancies promoted by in vivo administration of rapamycin in test mice.


Rapamycin Promotes Mouse 4T1 Tumor Metastasis that Can Be Reversed by a Dendritic Cell-Based Vaccine.

Lin TJ, Liang WM, Hsiao PW, M S P, Wei WC, Lin HT, Yin SY, Yang NS - PLoS ONE (2015)

Vaccination with SK-TCL-activated DCs effectively suppresses rapamycin-promoted metastasis of 4T1 cells after resection of the primary tumor.(A), Mice were injected subcutaneously with 4T1-Luc2 cells (5 × 105 cells/100 μl PBS/mouse) into the mammary fat pad at day 0. At 16 days post tumor cell implantation, orthotopic primary tumors were surgically resected. Test mice were administered with control (saline) or rapamycin by intraperitoneal injection, and subsequently with or without vaccination by SK-TCL-mDCs by intravenous injection at indicated time points. (B), Representative bioluminescent images of tumor-resected mice (n = 8) in each group shown at 4 weeks post tumor resection. The red signal represents the highest level on the colorimetric scale. Labelling with “D” in photograph denotes the mice that had died before 4 weeks post tumor resection. (C), Quantification of tumor metastasis by measuring luciferase activity in p/s/cm2/sr in mice revealed along the indicated time points (n = 8). (D), Survival of test mice after different treatments. Labelling symbol for the control mouse group was intentionally marked with relatively large open circles in order to allow a clear indication of data sets obtained from each experimental group. *, values of P < 0.05; **, P < 0.01, were obtained between the Rapa only and Rapa + SK-TCL + mDCs groups. Similar results were obtained from three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4591294&req=5

pone.0138335.g004: Vaccination with SK-TCL-activated DCs effectively suppresses rapamycin-promoted metastasis of 4T1 cells after resection of the primary tumor.(A), Mice were injected subcutaneously with 4T1-Luc2 cells (5 × 105 cells/100 μl PBS/mouse) into the mammary fat pad at day 0. At 16 days post tumor cell implantation, orthotopic primary tumors were surgically resected. Test mice were administered with control (saline) or rapamycin by intraperitoneal injection, and subsequently with or without vaccination by SK-TCL-mDCs by intravenous injection at indicated time points. (B), Representative bioluminescent images of tumor-resected mice (n = 8) in each group shown at 4 weeks post tumor resection. The red signal represents the highest level on the colorimetric scale. Labelling with “D” in photograph denotes the mice that had died before 4 weeks post tumor resection. (C), Quantification of tumor metastasis by measuring luciferase activity in p/s/cm2/sr in mice revealed along the indicated time points (n = 8). (D), Survival of test mice after different treatments. Labelling symbol for the control mouse group was intentionally marked with relatively large open circles in order to allow a clear indication of data sets obtained from each experimental group. *, values of P < 0.05; **, P < 0.01, were obtained between the Rapa only and Rapa + SK-TCL + mDCs groups. Similar results were obtained from three independent experiments.
Mentions: The results of Fig 3 further support the findings of our previous study using B16 cells [11] by once again demonstrating that shikonin-treated tumor cell lysate can be effectively employed to pulse and enhance mature dendritic cells (SK-TCL-mDC, Fig 3B and 3C) for therapeutic vaccination against metastasis of 4T1 tumors. As seen in Fig 2, we showed that in vivo treatment of mice with rapamycin promotes 4T1 tumor metastasis in our test mouse system. Therefore, next we were interested in whether rapamycin-promoted 4T1 cell metastasis after resection of the original primary tumor, could be suppressed by treatment with the SK-TCL-mDC cancer vaccine (Fig 4). In this experiment, an “anti-metastasis protocol” was designed in which the test DC vaccine was delivered on the very next day of tumor resection (Fig 4A). Bioluminescent imaging analysis of mice treated with control (saline) and different vaccine adjuvant formulations (mDCs only, SK-TCL-mDC, rapamycin only, and rapamycin + SK-TCL-mDC) were scored and analyzed. Consistent with the result of Fig 2, rapamycin treatment significantly increase the level of 4T1 tumor metastasis, as compared with the vehicle control treatment (P < 0.05) (Fig 4C), In contrast, SK-TCL-mDC treatment drastically reduced the tumor incidence and the rapamycin-promoted tumor metastasis, as evidenced by the lowest activity for metastasized tumors (Fig 4B and 4C). Tumor metastasis was suppressed by SK-TCL-mDC vaccination to a level of 62.5% at 2 months post tumor resection, as compared with the control group (P < 0.01) (Fig 4C). The level of anti-metastatic effect for rapamycin + SK-TCL-mDC treatment was similar to, i.e., not statistically different from, that of SK-TCL-mDC treatment (P = 0.94, n.s) (Fig 4C). The mean survival period of test mice with lung metastasis was 5, 12, 4 and 10 weeks for the control, SK-TCL-mDC, rapamycin, and rapamycin + SK-TCL-mDC group, respectively (Fig 4D). Co-treatment of rapamycin and SK-TCL-mDC vaccination (Rapa + SK-TCL-mDCs) significantly increased the life span of mice as compared to the vaccine-untreated (i.e., rapamycin alone) mice (P < 0.001), or the control group mice (i.e., untreated with DC vaccine and untreated with rapamycin) (Fig 4D). Together, these results clearly demonstrate that SK-TCL-mDC can effectively inhibit the initiation and progression of highly metastatic 4T1 malignancies promoted by in vivo administration of rapamycin in test mice.

Bottom Line: Suppression of tumor metastasis is a key strategy for successful cancer interventions.However, rapamycin also exhibits immunosuppressant effects and is hence used clinically as an organ transplantation drug.We hypothesized that the immunosuppressive activities of rapamycin might also negatively mediate host immunity, resulting in promotion of tumor metastasis.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Injury Prevention and Control, Taipei Medical University, Taipei, Taiwan, ROC; Department of Neurosurgery, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan, ROC; Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan, ROC; Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan, ROC; Taiwan International Graduate Program (TIGP), Molecular and Biological Agricultural Sciences Program, Academia Sinica, Taipei, Taiwan, ROC.

ABSTRACT
Suppression of tumor metastasis is a key strategy for successful cancer interventions. Previous studies indicated that rapamycin (sirolimus) may promote tumor regression activity or enhance immune response against tumor targets. However, rapamycin also exhibits immunosuppressant effects and is hence used clinically as an organ transplantation drug. We hypothesized that the immunosuppressive activities of rapamycin might also negatively mediate host immunity, resulting in promotion of tumor metastasis. In this study, the effects of rapamycin and phytochemical shikonin were investigated in vitro and in vivo in a 4T1 mouse mammary tumor model through quantitative assessment of immunogenic cell death (ICD), autophagy, tumor growth and metastasis. Tumor-bearing mice were immunized with test vaccines to monitor their effect on tumor metastasis. We found that intraperitoneal (ip) administration of rapamycin after a tumor-resection surgery drastically increased the metastatic activity of 4T1 tumors. Possible correlation of this finding to human cancers was suggested by epidemiological analysis of data from Taiwan's National Health Insurance Research Database (NHIRD). Since our previous studies showed that modified tumor cell lysate (TCL)-pulsed, dendritic cell (DC)-based cancer vaccines can effectively suppress metastasis in mouse tumor models, we assessed whether such vaccines may help offset this rapamycin-promoted metastasis. We observed that shikonin efficiently induced ICD of 4T1 cells in culture, and DC vaccines pulsed with shikonin-treated TCL (SK-TCL-DC) significantly suppressed rapamycin-enhanced metastasis and Treg cell expansion in test mice. In conclusion, rapamycin treatment in mice (and perhaps in humans) promotes metastasis and the effect may be offset by treatment with a DC-based cancer vaccine.

No MeSH data available.


Related in: MedlinePlus