Limits...
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

Shikonin treated tumor cell lysate- and autophagosome-activated DCs efficiently induce lymphocyte proliferation in vitro.(A), Effect of shikonin and rapamycin in treated 4T1 cells on expression of autophagosome markers. These organelle preparations were enriched, partially purified, and loaded onto DCs for vaccine preparation. Doxorubicin- and shikonin-treated 4T1 cells and the resultant-ICD derived TCLs were also used to pulse DCs for vaccine formulation. Effect of the resultant DC vaccines on in vitro proliferation of (B), Splenocytes and (C), CD8+ T cells, collected from syngeneic mice as responder cells, were analyzed. The p values, i.e., * P < 0.05; ** P < 0.01; *** P < 0.001; and n.s, no significance, were obtained between the two indicated test groups. Data represent the mean ± SE obtained from three independent experiments. (D), The secretory activity of IFN-γ in CD8+ T cells treated with various DC vaccine samples was evaluated be ELISA. (Rapa: Rapamycin; SK: Shikonin; AS: Autophagosome; TCL: Tumor cell lysate).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4591294&req=5

pone.0138335.g003: Shikonin treated tumor cell lysate- and autophagosome-activated DCs efficiently induce lymphocyte proliferation in vitro.(A), Effect of shikonin and rapamycin in treated 4T1 cells on expression of autophagosome markers. These organelle preparations were enriched, partially purified, and loaded onto DCs for vaccine preparation. Doxorubicin- and shikonin-treated 4T1 cells and the resultant-ICD derived TCLs were also used to pulse DCs for vaccine formulation. Effect of the resultant DC vaccines on in vitro proliferation of (B), Splenocytes and (C), CD8+ T cells, collected from syngeneic mice as responder cells, were analyzed. The p values, i.e., * P < 0.05; ** P < 0.01; *** P < 0.001; and n.s, no significance, were obtained between the two indicated test groups. Data represent the mean ± SE obtained from three independent experiments. (D), The secretory activity of IFN-γ in CD8+ T cells treated with various DC vaccine samples was evaluated be ELISA. (Rapa: Rapamycin; SK: Shikonin; AS: Autophagosome; TCL: Tumor cell lysate).

Mentions: We showed previously that a much enhanced immunogenicity and the efficacy of a SK-TCL-DC cancer vaccine were due to the ICD-inducing “adjuvant” effect of shikonin in the DC vaccine formulation.[11] To evaluate the potency of induction of autophagosomes by shikonin and rapamycin and their use in formulation as a vaccine adjuvant in our present 4T1 cell system, we stimulated and collected autophagosomes and loaded them onto DCs, and the effect of the resultant DCs on T-cell proliferation was assayed. As shown in Fig 3A, rapamycin and shikonin at 5 μM resulted in very effective induction of the autophagosome marker, LC3β-Ⅱ (Rapa-AS and SK-AS), in 4T1 cells, as compared to a control. Autophagosome and TCL preparations were hence isolated from 4T1 cells treated or not treated with rapamycin, doxorubicin or shikonin, and further tested for stimulation of splenocyte and CD8+ T cell proliferations. Doxorubicin-TCL loaded mature DCs exhibited the highest levels of stimulation of splenocyte proliferation; this was followed by SK-TCL-loaded mature DCs, shikonin-induced autophagosome (SK-AS) loaded mature DCs (SK-AS-mDCs), then rapamycin-induced autophagosome loaded mDCs (Rapa-AS, mDCs) (Fig 3B). Stimulation of CD8+ T cell proliferation among the treatment groups was in the following order, from highest level of proliferation to lowest level: SK-TCL-loaded mature DCs, shikonin- and then rapamycin—induced autophagosome (AS)-loaded mature DCs (Fig 3C). In addition, the secretory activity of IFN-γ in CD8+ T cells treated with various DC vaccine samples was evaluated by ELISA. Consistently, T cells pulsed with SK-TCL-mDCs expressed higher levels of IFN-γ (Fig 3D), and this was followed by stimulation with Dox-TCL-DCs. However, both SK-AS-mDCs and Rapa-AS-mDCs did not confer a significant effect on this activity of test CD8+ T cells. Our data hence demonstrate that the SK is an active phytochemical on induction of ICD activity in treated tumor cells and responsible for the subsequent tumor immunogenicity of cytotoxic T cells. These results were representative of three independent experiments and we, therefore, propose with confidence that in our system, rapamycin, unlike shikonin and doxorubicin, has very limited capability to augment the efficacy of our tumor cell-based cancer vaccines.


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)

Shikonin treated tumor cell lysate- and autophagosome-activated DCs efficiently induce lymphocyte proliferation in vitro.(A), Effect of shikonin and rapamycin in treated 4T1 cells on expression of autophagosome markers. These organelle preparations were enriched, partially purified, and loaded onto DCs for vaccine preparation. Doxorubicin- and shikonin-treated 4T1 cells and the resultant-ICD derived TCLs were also used to pulse DCs for vaccine formulation. Effect of the resultant DC vaccines on in vitro proliferation of (B), Splenocytes and (C), CD8+ T cells, collected from syngeneic mice as responder cells, were analyzed. The p values, i.e., * P < 0.05; ** P < 0.01; *** P < 0.001; and n.s, no significance, were obtained between the two indicated test groups. Data represent the mean ± SE obtained from three independent experiments. (D), The secretory activity of IFN-γ in CD8+ T cells treated with various DC vaccine samples was evaluated be ELISA. (Rapa: Rapamycin; SK: Shikonin; AS: Autophagosome; TCL: Tumor cell lysate).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591294&req=5

pone.0138335.g003: Shikonin treated tumor cell lysate- and autophagosome-activated DCs efficiently induce lymphocyte proliferation in vitro.(A), Effect of shikonin and rapamycin in treated 4T1 cells on expression of autophagosome markers. These organelle preparations were enriched, partially purified, and loaded onto DCs for vaccine preparation. Doxorubicin- and shikonin-treated 4T1 cells and the resultant-ICD derived TCLs were also used to pulse DCs for vaccine formulation. Effect of the resultant DC vaccines on in vitro proliferation of (B), Splenocytes and (C), CD8+ T cells, collected from syngeneic mice as responder cells, were analyzed. The p values, i.e., * P < 0.05; ** P < 0.01; *** P < 0.001; and n.s, no significance, were obtained between the two indicated test groups. Data represent the mean ± SE obtained from three independent experiments. (D), The secretory activity of IFN-γ in CD8+ T cells treated with various DC vaccine samples was evaluated be ELISA. (Rapa: Rapamycin; SK: Shikonin; AS: Autophagosome; TCL: Tumor cell lysate).
Mentions: We showed previously that a much enhanced immunogenicity and the efficacy of a SK-TCL-DC cancer vaccine were due to the ICD-inducing “adjuvant” effect of shikonin in the DC vaccine formulation.[11] To evaluate the potency of induction of autophagosomes by shikonin and rapamycin and their use in formulation as a vaccine adjuvant in our present 4T1 cell system, we stimulated and collected autophagosomes and loaded them onto DCs, and the effect of the resultant DCs on T-cell proliferation was assayed. As shown in Fig 3A, rapamycin and shikonin at 5 μM resulted in very effective induction of the autophagosome marker, LC3β-Ⅱ (Rapa-AS and SK-AS), in 4T1 cells, as compared to a control. Autophagosome and TCL preparations were hence isolated from 4T1 cells treated or not treated with rapamycin, doxorubicin or shikonin, and further tested for stimulation of splenocyte and CD8+ T cell proliferations. Doxorubicin-TCL loaded mature DCs exhibited the highest levels of stimulation of splenocyte proliferation; this was followed by SK-TCL-loaded mature DCs, shikonin-induced autophagosome (SK-AS) loaded mature DCs (SK-AS-mDCs), then rapamycin-induced autophagosome loaded mDCs (Rapa-AS, mDCs) (Fig 3B). Stimulation of CD8+ T cell proliferation among the treatment groups was in the following order, from highest level of proliferation to lowest level: SK-TCL-loaded mature DCs, shikonin- and then rapamycin—induced autophagosome (AS)-loaded mature DCs (Fig 3C). In addition, the secretory activity of IFN-γ in CD8+ T cells treated with various DC vaccine samples was evaluated by ELISA. Consistently, T cells pulsed with SK-TCL-mDCs expressed higher levels of IFN-γ (Fig 3D), and this was followed by stimulation with Dox-TCL-DCs. However, both SK-AS-mDCs and Rapa-AS-mDCs did not confer a significant effect on this activity of test CD8+ T cells. Our data hence demonstrate that the SK is an active phytochemical on induction of ICD activity in treated tumor cells and responsible for the subsequent tumor immunogenicity of cytotoxic T cells. These results were representative of three independent experiments and we, therefore, propose with confidence that in our system, rapamycin, unlike shikonin and doxorubicin, has very limited capability to augment the efficacy of our tumor cell-based cancer vaccines.

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