Mechanism and biological significance of constitutive expression of MGSA/GRO chemokines in malignant melanoma tumor progression

By reverse transcriptaseâ polymerase chain reaction, enzymelinked immunosorbent assay, and immunohistochemistry, MGSAâ α, â β, â γ, and CXCR2 mRNA expression and proteins are detected in 7 out of 10 human melanoma lesions. The biological consequence of constitutive expression of the MGSA/GRO chemokine in immortalized melanocytes was tested in SCID and nude mouse models. Continuous expression of MGSA/GROâ α, â β, or â γ in immortalized melanâ a mouse melanocytes results in nearly 100% tumor formation for each of the clones tested, whereas clones expressing only the neomycin resistance vector form tumors <10% of the time. Moreover, antibodies to the MGSA/GRO proteins slow or inhibit the formation of tumors in the SCID mouse model and block the angiogenic response to conditioned medium from the tumorâ producing clones. Transcription of the MGSA/ GRO chemokines is regulated by an enhancesomelike complex comprised of the nuclear factorâ κB (NFâ κB), HMG(I)Y, IUR, and Sp1 elements. In Hs294T melanoma cells the half life of the IκB protein is shortened in comparison to normal retinal epithelial cells, facilitating the endogenous nuclear localization of NFâ κB. We propose that this endogenous nuclear NFâ κB, working in concert with the 115â κDa IURâ binding factor, promotes constitutive expression of MGSA/GRO genes. J. Leukoc. Biol.62: 588â 597; 1997.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141559/1/jlb0588.pd

The tumorigenic and angiogenic effects of MGSA/GRO proteins in melanoma

Continuous expression of the MGSA/GROα, β, or γ chemokine bestows tumorâ forming capacity to the immortalized murine melanocyte cell line, melanâ a. The mechanism for this transformation is unclear, although both autocrine and paracrine processes are possible because melanâ a cells as well as endothelial cells express a low level of the receptor for this ligand. To further define the role of MGSA/GRO proteins in melanocyte transformation, two types of experiments were designed to neutralize the biological effects of MGSA/GRO in the transfected melanâ a clones: (1) the effect of neutralizing antiserum to MGSA/GRO proteins on melanâ a tumor growth was assessed; (2) the tumorâ forming capacity of melanâ a clones expressing ELR motifâ mutated forms of MGSA/GRO with compromised receptor affinity was compared to the tumorâ forming capacity of clones expressing wildâ type MGSA/GRO. These experiments revealed that SCID mice inoculated with MGSA/GROαâ or γâ expressing melanâ a cells and subsequently treated with antiserum to the respective chemokine exhibited decreased tumor growth. This reduction in tumor growth was accompanied by declining angiogenic activity in MGSA/GROγâ expressing tumors. Moreover, athymic nude mice injected with melanâ a cells expressing ELRâ mutant forms of MGSA/GROα exhibited markedly impaired tumorâ forming capacity compared with those mice injected with melanâ a clones expressing wildâ type MGSA/GRO. These data suggest that continuous expression of MGSA/GRO proteins may facilitate tumor growth by stimulating the growth of microvessels into the tumor (paracrine) and by affecting melanocyte growth (autocrine). J. Leukoc. Biol. 67: 53â 62; 2000.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142059/1/jlb0053.pd

Mechanism and biological significance of constitutive expression of MGSA/GRO chemokines in malignant melanoma tumor progression

By reverse transcriptaseâ polymerase chain reaction, enzymelinked immunosorbent assay, and immunohistochemistry, MGSAâ α, â β, â γ, and CXCR2 mRNA expression and proteins are detected in 7 out of 10 human melanoma lesions. The biological consequence of constitutive expression of the MGSA/GRO chemokine in immortalized melanocytes was tested in SCID and nude mouse models. Continuous expression of MGSA/GROâ α, â β, or â γ in immortalized melanâ a mouse melanocytes results in nearly 100% tumor formation for each of the clones tested, whereas clones expressing only the neomycin resistance vector form tumors <10% of the time. Moreover, antibodies to the MGSA/GRO proteins slow or inhibit the formation of tumors in the SCID mouse model and block the angiogenic response to conditioned medium from the tumorâ producing clones. Transcription of the MGSA/ GRO chemokines is regulated by an enhancesomelike complex comprised of the nuclear factorâ κB (NFâ κB), HMG(I)Y, IUR, and Sp1 elements. In Hs294T melanoma cells the half life of the IκB protein is shortened in comparison to normal retinal epithelial cells, facilitating the endogenous nuclear localization of NFâ κB. We propose that this endogenous nuclear NFâ κB, working in concert with the 115â κDa IURâ binding factor, promotes constitutive expression of MGSA/GRO genes. J. Leukoc. Biol.62: 588â 597; 1997.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141559/1/jlb0588.pd

Novel induction of CD40 expression by tumor cells with RAS/RAF/PI3K pathway inhibition augments response to checkpoint blockade

Abstract Background While immune checkpoint blockade (ICB) is the current first-line treatment for metastatic melanoma, it is effective for ~ 52% of patients and has dangerous side effects. The objective here was to identify the feasibility and mechanism of RAS/RAF/PI3K pathway inhibition in melanoma to sensitize tumors to ICB therapy. Methods Rigosertib (RGS) is a non-ATP-competitive small molecule RAS mimetic. RGS monotherapy or in combination therapy with ICB were investigated using immunocompetent mouse models of BRAFwt and BRAFmut melanoma and analyzed in reference to patient data. Results RGS treatment (300 mg/kg) was well tolerated in mice and resulted in ~ 50% inhibition of tumor growth as monotherapy and ~ 70% inhibition in combination with αPD1 + αCTLA4. RGS-induced tumor growth inhibition depends on CD40 upregulation in melanoma cells followed by immunogenic cell death, leading to enriched dendritic cells and activated T cells in the tumor microenvironment. The RGS-initiated tumor suppression was partially reversed by either knockdown of CD40 expression in melanoma cells or depletion of CD8+ cytotoxic T cells. Treatment with either dabrafenib and trametinib or with RGS, increased CD40+SOX10+ melanoma cells in the tumors of melanoma patients and patient-derived xenografts. High CD40 expression level correlates with beneficial T-cell responses and better survival in a TCGA dataset from melanoma patients. Expression of CD40 by melanoma cells is associated with therapeutic response to RAF/MEK inhibition and ICB. Conclusions Our data support the therapeutic use of RGS + αPD1 + αCTLA4 in RAS/RAF/PI3K pathway-activated melanomas and point to the need for clinical trials of RGS + ICB for melanoma patients who do not respond to ICB alone. Trial registration NCT01205815 (Sept 17, 2010). Graphical abstrac

Fine-Needle Aspiration-Based Patient-Derived Cancer Organoids

Patient-derived cancer organoids hold great potential to accurately model and predict therapeutic responses. Efficient organoid isolation methods that minimize post-collection manipulation of tissues would improve adaptability, accuracy, and applicability to both experimental and real-time clinical settings. Here we present a simple and minimally invasive fine-needle aspiration (FNA)-based organoid culture technique using a variety of tumor types including gastrointestinal, thyroid, melanoma, and kidney. This method isolates organoids directly from patients at the bedside or from resected tissues, requiring minimal tissue processing while preserving the histologic growth patterns and infiltrating immune cells. Finally, we illustrate diverse downstream applications of this technique including in vitro high-throughput chemotherapeutic screens, in situ immune cell characterization, and in vivo patient-derived xenografts. Thus, routine clinical FNA-based collection techniques represent an unappreciated substantial source of material that can be exploited to generate tumor organoids from a variety of tumor types for both discovery and clinical applications