Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Sep 1;6(4):451-464.
doi: 10.7150/ntno.75045. eCollection 2022.

Comparative effects of free doxorubicin, liposome encapsulated doxorubicin and liposome co-encapsulated alendronate and doxorubicin (PLAD) on the tumor immunologic milieu in a mouse fibrosarcoma model

Md Rakibul Islam  1 , Jalpa Patel  1 , Patricia Ines Back  1 , Hilary Shmeeda  2 , Konstantin Adamsky  3 , Hui Yang  4 , Carlos Alvarez  4 , Alberto A Gabizon  2   3 , Ninh M La-Beck  1   4
Affiliations

Comparative effects of free doxorubicin, liposome encapsulated doxorubicin and liposome co-encapsulated alendronate and doxorubicin (PLAD) on the tumor immunologic milieu in a mouse fibrosarcoma model

Md Rakibul Islam et al. Nanotheranostics. .

Abstract

Background: We have previously shown that alendronate, an amino-bisphosphonate, when reformulated in liposomes, can significantly enhance the efficacy of cytotoxic chemotherapies and help remodel the immunosuppressive tumor microenvironment towards an immune-permissive milieu resulting in increased anticancer efficacy. In addition, we have previously shown that the strong metal-chelating properties of alendronate can be exploited for nuclear imaging of liposomal biodistribution. To further improve anticancer efficacy, a pegylated liposome formulation co-encapsulating alendronate and doxorubicin (PLAD) has been developed. In this study, we examined the effects of PLAD on the tumor immunologic milieu in a mouse fibrosarcoma model in which the tumor microenvironment is heavily infiltrated with tumor-associated macrophages (TAM) that are associated with poor prognosis and treatment resistance. Methods: Doxorubicin biodistribution, characterization of the tumor immunologic milieu, cellular doxorubicin uptake, and tumor growth studies were performed in Balb/c mice bearing subcutaneously implanted WEHI-164 fibrosarcoma cells treated intravenously with PLAD, pegylated liposomal doxorubicin (PLD), free doxorubicin, or vehicle. Results: PLAD delivery resulted in a high level of tumor doxorubicin that was 20 to 30-fold greater than in free doxorubicin treated mice, and non-significantly higher than in PLD treated mice. PLAD also resulted in increased uptake in spleen and slightly lower plasma levels as compared to PLD. Importantly, our results showed that PLAD, and to a lesser extent PLD, shifted cellular drug uptake to TAM and to monocytic myeloid-derived suppressor cells (MDSC), while there was no drug uptake in neutrophilic MDSC or lymphoid cells. Free doxorubicin cellular drug uptake was below detectable levels. PLAD, and to a lesser extent PLD, also induced significant changes in number and functionality of tumor-infiltrating TAM, MDSC, Treg, NKT, and NK cells that are consistent with enhanced antitumor immune responses in the tumor microenvironment. In contrast, free doxorubicin induced moderate changes in the tumor microenvironment that could promote (decreased Treg) or be detrimental to antitumor immune responses (decreased M1 TAM and NK cells). These immune modulatory effects are reflected in the therapeutic study which showed that PLAD and PLD inhibited tumor growth and significantly prolonged survival, while free doxorubicin showed little or no anticancer activity. Conclusion: We show that liposomal delivery of doxorubicin not only alters pharmacokinetics, but also dramatically changes the immune modulatory activity of the drug cargo. In addition, our data support that the PLAD nanotheranostic platform further enhances some immune changes that may act in synergy with its cytotoxic chemotherapy effects.

Keywords: alendronate; bisphosphonate; chemotherapy; doxorubicin; fibrosarcoma; immunotherapy; nanomedicine; tumor-associated macrophages.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Experiment Schema. (A) Pictograms and (B) cryoTEM images of PLD and PLAD liposomes. (C) Overview of the three in vivo studies.
Figure 2
Figure 2
PLAD, PLD and F-Dox biodistribution in WEHI-164 tumor bearing mice at 72 h (PLAD, PLD) and 2 h (F-Dox) post-dose. Data are mean with SEM; n=7 for PLD and PLAD, n=5 for F-Dox. ANOVA with Tukey's test (A, B, D) or unpaired t-test (C); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. F-Dox: free doxorubicin, PLD: pegylated liposomal doxorubicin, PLAD: pegylated liposomal alendronate-doxorubicin.
Figure 3
Figure 3
PLAD and PLD reduced TAM and increased monocytic MDSC in the tumor microenvironment. (A) Gating strategy for TAM, mMDSC, and nMDSC. (B) PLAD and PLD significantly decreased the number of TAM and (C) increased mMDSCs in tumors. PLAD also increased nMDSC but the other treatments did not. Representative FACS plots are shown. Data are mean with SEM, n=9 for PLAD, PLD and F-Dox, n=5 for vehicle; ANOVA with Tukey's test; **p<0.01, ***p<0.001, and ****p<0.0001. F-Dox: free doxorubicin, PLD: pegylated liposomal doxorubicin, PLAD: pegylated liposomal alendronate doxorubicin, TAM: tumor associated macrophages, mMDSC: monocytic myeloid derived suppressor cells, nMDSC: neutrophilic myeloid derived suppressor cells.
Figure 4
Figure 4
Effects of doxorubicin on TAM polarization depends on liposomal drug delivery and alendronate co-encapsulation. (A) TAM polarization was determined in the TAM population from Figure 3A. (B) All treatments significantly increased non-polarized M0 TAM. Free doxorubicin treatment decreased M1 TAM, while PLD and PLAD had no effects on this population. M2 polarized TAM were decreased in PLD and PLAD groups with the greatest effect in PLAD, although the difference between PLD and PLAD was not significant. All treatments also decreased M1-M2 TAM, a population that expresses both M1 and M2 markers. (C-D) PLAD significantly increased M1/M2 ratio compared to F-Dox. Representative FACS plots are shown. Data are mean with SEM, n=9 for PLAD, PLD and F-Dox, n=5 for vehicle; ANOVA with Tukey's test; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. F-Dox: free doxorubicin, PLD: pegylated liposomal doxorubicin, PLAD: pegylated liposomal alendronate doxorubicin, TAM: tumor associated macrophages.
Figure 5
Figure 5
PLAD increased antigen cross presenting dendritic cell infiltration and NK cell activation in tumors. (A-B) Antigen cross presenting dendritic cells (DC1) and conventional dendritic cells (DC2) were gated from the CD11c+ population in Figure 3A. PLAD increased tumor infiltration of DC1, while DC2 infiltration was not affected by any treatment. (C) Gating strategy for NK cells. (D) Although NK cell infiltration in the tumor microenvironment was not affected, (E) PLD and PLAD increased the proportion of activated NK cells. Representative FACS plots are shown. Data are mean with SEM, n=9 for PLAD, PLD and F-Dox, n=5 for vehicle; ANOVA with Tukey's test; *p<0.05, **p<0.01, and ****p<0.0001. F-Dox: free doxorubicin, PLD: pegylated liposomal doxorubicin, PLAD: pegylated liposomal alendronate doxorubicin, DC: dendritic cells, NK: natural killer cells.
Figure 6
Figure 6
PLAD decreased T regulatory cells and increased activated NKT cells in tumors. (A-B) Helper and cytotoxic T cells were identified from the CD3+ population in Figure 5C. There were no significant differences in the total tumor infiltrating T cell population. However, further inspection showed that there was a significant decrease in helper T cells in PLAD treated animals. There was no significant impact on cytotoxic T cells for any treatment. (C) PLAD showed a decreased CD4/CD8 ratio, but it was not statistically significant. (D) T regulatory cells were gated from live cells (Supplemental Figure S4). (E) PLAD, PLD, and F-Dox significantly decreased the infiltration of regulatory T cells in tumors. (F) PLAD and PLD also increased the activation of NKT cells in tumors. Representative FACS plots are shown. Data are mean with SEM, n=9 for PLAD, PLD, and F-Dox, n=5 for vehicle; ANOVA with Tukey's test; *p<0.05, **p<0.01, and ***p<0.001. F-Dox: free doxorubicin, PLD: pegylated liposomal doxorubicin, PLAD: pegylated liposomal alendronate doxorubicin, NKT: natural killer T-cells.
Figure 7
Figure 7
Liposomal drug delivery significantly increases internalization of doxorubicin by TAM and mMDSC. (A) Gating strategy for doxorubicin fluorescence. (B) Doxorubicin uptake in TAM, non-myeloid leukocytes (i.e., lymphocytes), and non-leukocytes (i.e., tumor and stromal cells). (C) Doxorubicin uptake in TAM by polarization state and treatment. (D) Doxorubicin uptake in mMDSC and nMDSC. Data are mean with SEM, n=9 for PLAD, PLD and F-Dox, n=5 for vehicle; ANOVA with Tukey's test; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. F-Dox: free doxorubicin, PLD: pegylated liposomal doxorubicin, PLAD: pegylated liposomal alendronate doxorubicin, TAM: tumor associated macrophages, mMDSC: monocytic myeloid derived suppressor cells, nMDSC: neutrophilic myeloid derived suppressor cells.
Figure 8
Figure 8
Doxorubicin accumulation in the tumor by fluorescence microscopy. (A) Doxorubicin fluorescence in tumor sections showed higher uptake in tumors from PLAD group. Each point is one slide image, 4-11 images/tumor, 23 total animals (PLAD n=7, PLD n=8, F-Dox n=5, and vehicle n=3), bars represent group mean; ANOVA and Dunnett's test; *p<0.05, **p<0.01. (B) Representative images shown. F-Dox: free doxorubicin, PLD: pegylated liposomal doxorubicin, PLAD: pegylated liposomal alendronate doxorubicin; DAPI is a nuclear dye.
Figure 9
Figure 9
PLAD and PLD showed superior antitumor efficacy over free doxorubicin in the WEHI-164 fibrosarcoma model. (A) Tumor growth curves showing group mean with SEM; ANOVA with Dunnett's test; *versus F-Dox, p = 0.0267 and 0.0455, respectively, for PLAD and PLD. (B) Kaplan-meier curves for survival endpoint of 5-fold tumor growth; Log-rank tests. PLD, PLAD, and F-Dox, n=9 each group; vehicle n=6. F-Dox: free doxorubicin, PLD: pegylated liposomal doxorubicin, PLAD: pegylated liposomal alendronate doxorubicin.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Edwards TM, Duran MS, Meeker TM. Congenital Infantile Fibrosarcoma in the Premature Infant. Adv Neonatal Care. 2017;17:440–50. - PubMed
    1. Folpe AL. Fibrosarcoma: a review and update. Histopathology. 2014;64:12–25. - PubMed
    1. Das B, Jain N, Mallick B. piR-39980 mediates doxorubicin resistance in fibrosarcoma by regulating drug accumulation and DNA repair. Commun Biol. 2021;4:1312. - PMC - PubMed
    1. Hoefkens F, Dehandschutter C, Somville J, Meijnders P, Van Gestel D. Soft tissue sarcoma of the extremities: pending questions on surgery and radiotherapy. Radiat Oncol. 2016;11:136. - PMC - PubMed
    1. Augsburger D, Nelson PJ, Kalinski T, Udelnow A, Knosel T, Hofstetter M. et al. Current diagnostics and treatment of fibrosarcoma -perspectives for future therapeutic targets and strategies. Oncotarget. 2017;8:104638–53. - PMC - PubMed

Publication types