The frequent development of drug resistance to targeted therapies in cancer patients has stimulated interest in strategies counteracting resistance. Combining immunotherapies with targeted therapies is one such strategy. In this context, we asked whether human NK cells can target melanoma cells that have acquired resistance to selective inhibitors targeting activating mutants of the B‐Raf kinase (BRAF inhibitors, BRAFi). We generated drug‐resistant cell variants in vitro from human BRAF‐mutant melanoma cell lines MEL‐HO, COLO‐38, SK‐MEL‐37, 1520 and from primary melanoma cells freshly isolated from two patients. All drug‐resistant cell variants remained susceptible to lysis by IL‐2‐activated NK cells; and two BRAFi‐resistant lines (BRAFi‐R) became significantly more susceptible to NK‐cell lysis than their parental lines. This was associated with significant HLA class I antigen downregulation and PD‐L1 upregulation on the drug‐resistant lines. Although blocking HLA class I enhanced the extent of lysis of both BRAFi‐R and parental cells to NK‐cell‐mediated lysis, antibody‐mediated inhibition of PD1–PD‐L1 interactions had no detectable effect. HLA class I antigen expression on BRAFi‐R melanoma variants thus appears to play a major role in their susceptibility to NK‐cell cytotoxicity. These findings suggest that NK‐cell‐based immunotherapy may be a viable approach to treat melanoma patients with acquired resistance to BRAF inhibitors.
40-50% of patients with melanoma are identified with BRAF mutations. About 80% of the BRAF mutations carry the V600E mutation. These patients are treated with BRAF inhibitors alone or in combination with other cell signaling pathway inhibitors. However, a relapse of the disease is observed within 6-8 months after the commencement of the therapy. Some studies hint that NK cell based therapy can be effective in controlling melanoma. In this study multidisciplinary approaches were tested to understand the mechanism of Vemurafenib resistance by a BRAFV600E melanoma. Vemurafenib is a BRAFV600E inhibitor. Vemurafenib resistant melanoma cell lines were raised from the sensitive cell lines by drug treatment till 80-90% of cells showed resistance. Both the resistant and sensitive cell lines were tested in a NK cell cytotoxicity assay in vitro by standard 51Cr-release assay. The Vemurafenib resistant cell lines were found to have a reduced sensitivity to NK cell killing. Though there was no significant difference in expression of NK cell activating ligands on the surface between the resistant and sensitive cell lines, there was a tendency of higher expression of MHC class I on Vemurafenib resistant cell lines. BRAF exon15 sequencing shows no difference between Vemurafenib resistant and sensitive cell types. Experiments are in progress to understand the mechanism(s) of the reduced sensitivity of the Vemurafenib resistant cell lines to NK cell killing.
Monoclonal antibody‐based immunotherapy is a promising treatment for several diseases such as cancer and virus infection. However, due to its highly selective target, drug testing using animal models or classical 2D human cell cultures are not sufficient to predict the drug effects on patients. New 3D organotypic models that employ 3D cell culture of human cells within a microfluidic device allow detailed characterization of critical biological interactions. In this project, we describe a new 3D cell culture model that allows the creation of in‐vitro human perfusable vasculature containing hollow spaces for the insertion of tumor spheroids. A polydimethylsiloxane device with several vertical holes on top of the gel channel is used to create the host microvascular endothelial (HOME) network (Fig.1A). Endothelial cells and fibroblasts create perfusable vascular networks (Fig. 1B). Tumor spheroids are formed by coculturing MDA‐MB‐468 tumor cells (T), fibroblasts (F), and non‐polarized (M0) or M2 macrophages, and denoted by TFM0 and TFM2 respectively (Fig. 1C). One day after inserting a tumor spheroid in collagen into the HOME networks through the hole, freshly‐isolated monocytes are perfused into the vascular networks by luminal flows. We observe that after 2 days, the majority of monocytes remain either within the vasculature or extravasated but stay inside the fibrin gel outside of the hole containing the tumor spheroid (Fig. 1Di). On day 2, interstitial flows from the tumor spheroid cause monocyte migration from the vascular network side into the hole containing the tumor spheroid (figure 1Dii). When a TFM2 spheroid is inserted into the HOME networks, it recruits more monocytes than the TFM0 spheroid (Fig. Ei and Eii). Recruitment % is the percentage of monocytes that leave the blood vessel and migrate into the hole among the total number of monocytes in 3mmx3mm region of interest around the hole. Monocytes are recruited better by the TFM2 spheroid than the TFM0 spheroid (Fig. 1Gi). These results showed that interstitial flows from the tumor spheroid to the vascular networks and macrophage polarization are essential for monocyte recruitment. Multi‐specific antibody UniTI102 developed by Elstar Therapeutics (MA, USA) neutralizes chemokine CCL2 to block monocyte recruitment. To characterize the response to the drug UniTI102, we compared devices having a TFM0 spheroid that are untreated (Fig. 1D) or treated with UniTI102 (Fig. 1F) and observed that less monocytes has migrated into the hole in the presence of UniTI102 at 100nM. Quantitative data also showed that less monocytes are recruited when devices are treated with 100nM UniTI102 than 100nM IgG (Fig. 1Gii).
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