Camptothesome Potentiates PD-L1 Immune Checkpoint Blockade for Improved Metastatic Triple-Negative Breast Cancer Immunochemotherapy

Abstract

In this study, we focus on investigating the therapeutic effects of camptothesome on treating metastatic triple-negative breast cancer (TNBC). We elucidate that camptothesome elicited stronger immunogenic cell death (ICD) compared to free camptothecin (CPT) and Onivyde in 4T1 TNBC cells. In addition, camptothesome is mainly internalized by the 4T1 and MDA-MB-231 cells through clathrin-mediated endocytosis based on the results of flow cytometry. Through real-time Lago optical imaging, camptothesome shows excellent tumor-targeting efficiency in orthotopic TNBC tumors. We demonstrate that camptothesome can upregulate programmed death-ligand 1 (PD-L1) in 4T1 tumors in an interferon gamma (IFN-γ)-dependent manner. Furthermore, the anti-TNBC efficacy studies reveal that camptothesome is superior to Onivyde and markedly potentiates PD-L1 immune checkpoint blockade therapy with complete lung metastasis remission in an orthotopic 4T1-Luc2 tumor model. This combination therapy eliciting robust cytotoxic T lymphocytes (CTL) response via boosting tumor-infiltrating cluster of differentiation 8 (CD8), calreticulin (CRT), high mobility group box 1 protein (HMGB-1), low-density lipoprotein receptor-related protein 1 (LRP1), IFN-γ, and granzyme B. Our work corroborates the promise of camptothesome in favorably modulating tumor immune microenvironment via inducing ICD to fortify the PD-L1 checkpoint blockade therapy for improved treatment of intractable TNBC.

Keywords: camptothecin; camptothesome; immune checkpoint blockade; immunogenic cell death; nanovesicle; triple-negative breast cancer.

Figure 1.. Camptothesome induces ICD response in TNBC.

(a) Flow cytometry investigation of the apoptotic and necrotic 4T1 cells by FITC Annexin V and propidium iodide (PI) after 20 h of treatment with free CPT, Onivyde^® and Camptothesome at 4 μM or 15 μM of equivalent (eq.) CPT. Lipo-SM/Chol was used at the eq. SM as used in Camptothecome. (b) Frequency of early apoptotic (Annexin V⁺ PI⁻) and necrotic/late apoptotic (Annexin V⁺ PI⁺) 4T1 cells. (c) Flow cytometry determination of the calreticulin expression on 4T1 cells induced by free CPT, Onivyde^® or Camptothesome for 20 h at 4 μM or 15 μM of CPT or irinotecan, respectively. (d) Normalized calreticulin expression ratio as compared to no treatment control. (e, f) The released ATP (e) and HMGB-1 (f) concentrations in the media of samples from (d), respectively. Data are expressed as mean ± s.d. (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2.. Intracellular uptake pathway of Camptothesome.

Mouse TNBC cells 4T1 (a, b) and human TNBC cell line MDA-MB-231 (c, d) were pretreated with various endocytotic inhibitors for 0.5 h then incubated with DSPE-Cy5.5-labeled Camptothesome (50 μg CPT/mL) in the presence of the inhibitors for additional 2 h. (a, c) Representative flow cytometry histograms of Cy5.5/Camptothesome. (b, d) Cellular uptake percentage rate of Cy5.5/Camptothesome analyzed by flow cytometry. Data in b and d are expressed as mean ± s.d. (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001 vs control.

Figure 3.. Biodistribution of Camptothesome.

(a) Lago optical imaging in orthotopic 4T1-Luc2 tumor-bearing BALB/c mice intravenously administered by free DSPE-Cy5.5 and DSPE-Cy5.5-labeled Camptothesome (20 mg CPT/kg) once (n = 3 mice; tumors: ~400 mm³). (b) Representative ex vivo imaging of different organs at 48 h after intravenous administration.

Figure 4.. PD-L1 was upregulated by Camptothesome in TNBC tumor.

Orthotopic 4T1-Luc2 tumor-bearing mice (n = 3; tumors: ~200 mm³) received a single intravenous injection of 5% Dextrose, Onivyde^® (33.6 mg irinotecan/kg) or Camptothesome at equivalent 20 mg CPT/kg with or without α-IFN-γ (intraperitoneally, 200 μg/mouse/3 days, 3 times). 10 days post treatment, mice were sacrificed, and the tumors were collected and divided into 3 pieces for ELASA, flow cytometry and IHC staining. (a) IFN-γ levels in tumor tissues measured by ELISA. (b) PD-L1 expression in tumor tissues were evaluated by flow cytometry through gating on tumor cells (CD45⁻ cells). (c) Mean fluorescence intensity (MFI) quantification of PD-L1 expression on tumor cells from flow cytometry. Data in a and c are expressed as mean ± s.d. (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, ****P < 0.0001.

Figure 5.. Bolstered therapeutic efficacy in orthotopic 4T1-Luc2 mouse model by combining PD-L1 checkpoint blockade.

On day 17, when the primary tumors reached ~200 mm³, mice (n = 6) were intravenously administered with Camptothesome, Onivyde^® (33.6 mg irinotecan/kg) alone at equivalent 20 mg CPT/kg or combined with α-PD-L1 (intraperitoneally, 100 μg/mouse/3 days, 3 times) on day 17 and 27. (a) Tumor growth curves. (b) Body weight monitoring. (c) Primary tumor weight on day 37. (d) Tumor-bearing mice images (left portion) and representative ex vivo lung metastasis Lago bioluminescence imaging (right portion) were taken on day 37. Data in a, b, c, are expressed as mean ± s.d. (n = 6). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 6.. Anti-TNBC immune responses.

Representative IHC staining for CD8, Calreticulin, HMGB1, LRP1, IFN-γ, Granzyme B, and respective semi-quantitative analysis independent tumors from Fig. 5, scale bar = 100 μm. Data in right panel are expressed as mean ± s.d. (n = 6). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Scheme 1.

Synthesis of Camptothesome