Clone M-3; Cloudman S91 melanoma

Intratumoral Treatment of Melanoma Tumors with Large Surface Area Microparticle Paclitaxel and Synergy with Immune Checkpoint Inhibition

Melanoma treatment often requires a combination of therapies. Intratumoral drug delivery systems offer a way to directly target tumors. Maulhardt et al. evaluated the effects of intratumoral large surface area microparticle paclitaxel (LSAM-PTX) alone and in combination with systemic anti-PD-1 therapy in a murine melanoma model.

The original melanocyte cell line Clone M-3 [Cloudman S91 melanoma] was passaged once in vivo, resulting in clone M3(Z1) and implanted into the left mammary fat pad. Ten days after tumor implant, animals were block-randomized into five groups (n=8/group) with group mean TV = 56.8 mm3 (range 42.8–66.3 mm3) and treatments were initiated. On Day 20, the IT LSAM-PTX-treated and IP anti-mPD-1-treated groups showed noticeable nonsignificant inhibition of TV compared to the vehicle-isotype control group and TV was further reduced in the group treated with the combination of IT LSAM-PTX + IP anti-mPD-1 (Fig. 1a). When TV and tumor weight was measured following necropsy on Day 20, the combined treatment of IT LSAM-PTX and IP anti-mPD-1 resulted in significant (p < 0.05) reduction in both TV (Fig. 1b) and tumor weight compared to control-treated animals.

TβRI-TβRII-Fc Chimeric Receptor Efficiently Suppresses TGF-β Signals in Melanoma Cells

Melanoma is an aggressive cancer originating from melanocytes. Transforming growth factor‑β (TGF‑β) promotes melanoma progression by inducing the epithelial‑mesenchymal transition (EMT) and creating a tumor-favorable environment. Three TGF‑β isoforms signal through SMAD pathways, therefore, all TGF-β isoforms should be targeted. Kodama et al. validated the activity of Fc chimeric receptors for TGF-β isoforms in melanoma.

In this study, B16 melanoma cells were used. 293T cells were transfected with vectors expressing Control-Fc, TβRII-Fc, or TβRI-TβRII-Fc chimeric receptors and the accumulation of these chimeric proteins in the conditioned media was validated by immunoblotting. B16 cells were incubated with the conditioned media of 293T cells expressing Control-Fc, TβRII-Fc, or TβRI-TβRII-Fc chimeric receptors in the presence or absence of TGF-β1, -β2, or -β3. Expression of the TGF-β-responsive genes TMEPAI and PAI-1 were evaluated by RT-qPCR. TGF-β isoforms upregulated TMEPAI (Fig. 2A) and PAI-1 (Fig. 2B) expression. Treatment of cells with TβRI kinase inhibitor SB431542 decreased the expression of these genes to the background level (Fig. 2). As expected, Control-Fc did not abrogate TGF-β-induced TMEPAI and PAI-1 expression. TβRII-Fc blocked TGF-β1 and TGF-β3 but not TGF-β2-induced expression of both genes (Fig. 2). In contrast, TβRI-TβRII-Fc significantly inhibited expression of both TMEPAI and PAI-1 genes induced by all three TGF-β isoforms, demonstrating its capacity to block TGF-β signals in melanoma model. To confirm these results, the same experiments were repeated in another melanoma cell line Clone M3. Clone M3 cells were also responsive to TGF-β isoforms, and TGF-β treatment increased expression of TMEPAI (Fig. 3A) and PAI-1 (Fig. 3B). Incubation with TβRI-TβRII-Fc decreased TMEPAI and PAI-1 expression induced by all three TGF-βs in comparison with Control-Fc, demonstrating that TβRI-TβRII-Fc abrogated TGF-β signals in two melanoma cell types.