Nanoparticle-Mediated Trapping of Wnt Family Member 5A in Tumor Microenvironments Enhances Immunotherapy for B-Raf Proto-Oncogene Mutant Melanoma

Abstract

Development of an effective treatment against advanced tumors remains a major challenge for cancer immunotherapy. Approximately 50% of human melanoma is driven by B-Raf proto-oncogene mutation (BRAF mutant). Tumors with such mutation are desmoplastic, highly immunosuppressive, and often resistant to immune checkpoint therapies. We have shown that immunotherapy mediated by low-dose doxorubicin-induced immunogenic cell death was only partially effective for this type of tumor and not effective in long-term inhibition of tumor progression. Wnt family member 5A (Wnt5a), a signaling protein highly produced by BRAF mutant melanoma cells, has been implicated in inducing dendritic cell tolerance and tumor fibrosis, thus hindering effective antigen presentation and T-cell infiltration. We hypothesized that Wnt5a is a key molecule controlling the immunosuppressive tumor microenvironment in metastatic melanoma. Accordingly, we have designed and generated a trimeric trap protein, containing the extracellular domain of Fizzled 7 receptor that binds Wnt5a with a K_d ∼ 278 nM. Plasmid DNA encoding for the Wnt5a trap was delivered to the tumor by using cationic lipid-protamine-DNA nanoparticles. Expression of Wnt5a trap in the tumor, although transient, was greater than that of any other major organs including liver, resulting in a significant reduction of the Wnt5a level in the tumor microenvironment without systematic toxicity. Significantly, combination of Wnt5a trapping and low-dose doxorubicin showed great tumor growth inhibition and host survival prolongation. Our findings indicated that efficient local Wnt5a trapping significantly remodeled the immunosuppressive tumor microenvironment to facilitate immunogenic cell-death-mediated immunotherapy.

Keywords: B-Raf proto-oncogene mutant melanoma; Wnt family member 5A; immune trap; immunogenic cell death; nanoparticle; tumor microenvironment.

Figure 1. ICD induced by low dose DOX

(A) MTT result on BPD6 cell line in vitro. n = 5. (B) Fluorescence imaging detecting ICD markers: CRT and HMGB1 on BPD6 cells treated with low dose of DOX. Cell nuclei were stained with Hoechst 33432. Scale bar indicates 10 µm. (C) ELISpot test depicting IFN-γ secreted by re-stimulated splenocytes of mice treated with or without low dose DOX. n = 3. (D) Flow cytometry analysis shows ICD induced increase in: intra-tumoral inflammatory cells (CD8⁺CD45⁺) and activation of CD8⁺ T cell within TME. n = 3. (E) HMGB1 and CRT immunofluorescence staining in tumors slide sections, treated with or without low dose of DOX. n = 3. Scale bar indicates 300 µm. (F) Mean days of mouse survival in PBS and low dose DOX treated groups. n = 8–10. Data present mean ± SE. n.s.: p > 0.05, **: p < 0. 01.

Figure 2. Wnt5a is a key molecule controlling the immunosuppressive desmoplastic TME

(A) Figure legend depicting Wnt5a functions on both DCs and Fibroblasts within TME. Wnt5a is highly expressed among BRAF-mutant melanoma, compared to BRAF-wild-type, in both clinical (panel B, TCGA database, n = 368) and murine samples (panel C, Western blot, n = 3). (D) High level of Wnt5a correlates with poorer patient overall survival. n = 29. (E) Masson’s trichrome staining illustrating BRAF-mutant melanoma with desmoplastic collagen-rich TME (black arrows), compared to wild-type in both human and mouse specimens. Scale bar indicates 300 µm. n = 3. *: p < 0.05, **: p < 0. 01.

Figure 3. Local distribution and expression of Wnt5a trap

(A and B) Development and characterization of Wnt5a Trap protein. (A) SDS-PAGE of the Wnt5a trap in the presence (lane 2) and absence (lane 1) of reducing agent DTT. (B) The binding affinity between Wnt5a and FZD7-based trap measured by MST. (C) Bio-distribution of DiI-loaded LPD NPs among tumor and organs. n = 3. (D) Expressions of His-tagged Wnt5a trap in different organs were quantified by ELISA and compared with the injection of free trap protein. n = 5. (E) Fluorescent imaging depicting effective local Wnt5a trapping in tumor slide sections. Numbers indicate Wnt5a expression (yellow). Scale bar indicates 300 µm. (F) Mean days of mouse survival. n = 8–10. Data present mean ± SE. n.s.: p > 0.05, **: p < 0. 01.

Figure 4. Combination therapy significantly inhibited tumor progression

(A) Tumor inhibition of combination therapy, compared with untreated or monotherapy groups. n = 8. (B) Long term survival monitored over two months. n = 8–10. (C) Tumor bearing mice were pretreated with 3 daily injections of anti-CD8 and/or anti-CD4 antibody (300 µg/mice) to deplete the CD8⁺ and/or CD4⁺ T cells in vivo. Isotype IgG was used as control. The efficacy of combination therapy with or without different T cell depletion was compared by monitoring tumor burden after treatment cycles. n = 5. (D) TUNEL assay depicting apoptotic region within tumor slide sections. n = 3. (E) Masson’s trichrome staining depicting collagen distribution within TME. n = 3. Scale bars indicate 300 µm. *: p < 0.05, **: p < 0. 01, ***: p < 0. 001.

Figure 5. Remodeling of tumor microenvironment

(A) Flow cytometry analysis of immune functioning cells within TME. n = 3. (B) ELIspot assay depicting IFN-γ production under different treatments. n = 3. (C) RT-PCR analysis of both pro-inflammatory and anti-inflammatory cytokines within TME. n = 6. *: p < 0.05, **: p < 0. 01, ***: p < 0. 001.

Figure 6. Combination therapy demonstrated long-lasting overall immune response

(A) Immunofluorescent staining for memory CD4 T cells within TME. n = 3. Scale bar indicates 100 µm. (B) and (C) Flow cytometry analysis of T cells and DCs functions within draining LNs. n = 3. (D) Immunofluorescent staining for memory CD8 T cells within TME. n = 3. Scale bar indicates 100 µm. (E) In vitro CD4⁺ T cell proliferation assay depicting division of antigen-reactive cells (Q1) between groups. n = 3. *: p < 0.05, **: p < 0. 01.

Figure 7. Toxicity evaluation of therapies

(A) Mice body weight changes under tumor inhibition study. (B) Whole cell counts and serum biochemical marker analysis of tumor bearing mice. Fresh whole blood and serum were collected at endpoint of study. Non-tumored mice were also examined as control. n = 5. n.s.: p > 0.05.