An important component of research using animal models is ensuring rigor and reproducibility.,This study was prompted after two experimenters performing virtually identical studies obtained different results when syngeneic B78 murine melanoma cells were implanted into the skin overlying the flank and treated with an in situ vaccine (ISV) immunotherapy.,Although both experimenters thought they were using identical technique, we determined that one was implanting the tumors intradermally (ID) and the other was implanting them subcutaneously (SC).,Though the baseline in vivo immunogenicity of tumors can depend on depth of their implantation, the response to immunotherapy as a function of tumor depth, particularly in immunologically ‘cold’ tumors, has not been well studied.,The goal of this study was to evaluate the difference in growth kinetics and response to immunotherapy between identically sized melanoma tumors following ID versus SC implantation.,We injected C57BL/6 mice with syngeneic B78 melanoma cells either ID or SC in the flank.,When tumors reached 190-230 mm3, they were grouped into a ‘wave’ and treated with our previously published ISV regimen (12 Gy local external beam radiation and intratumoral hu14.18-IL2 immunocytokine).,Physical examination demonstrated that ID-implanted tumors were mobile on palpation, while SC-implanted tumors became fixed to the underlying fascia.,Histologic examination identified a critical fascial layer, the panniculus carnosus, which separated ID and SC tumors.,SC tumors reached the target tumor volume significantly faster compared with ID tumors.,Most ID tumors exhibited either partial or complete response to this immunotherapy, whereas most SC tumors did not.,Further, the ‘mobile’ or ‘fixed’ phenotype of tumors predicted response to therapy, regardless of intended implantation depth.,These findings were then extended to additional immunotherapy regimens in four separate tumor models.,These data indicate that the physical ‘fixed’ versus ‘mobile’ characterization of the tumors may be one simple method of ensuring homogeneity among implanted tumors prior to initiation of treatment.,Overall, this short report demonstrates that small differences in depth of tumor implantation can translate to differences in response to immunotherapy, and proposes a simple physical examination technique to ensure consistent tumor depth when conducting implantable tumor immunotherapy experiments.
Anthracycline chemotherapeutics, e.g. doxorubicin and daunorubicin, are active against a broad spectrum of cancers.,Their cytotoxicity is mainly attributed to DNA intercalation, interference with topoisomerase activity, and induction of double-stranded DNA breaks.,Since modification of anthracyclines can profoundly affect their pharmacological properties we attempted to elucidate the mechanism of action, and identify possible molecular targets, of bis-anthracycline WP760 which previously demonstrated anti-melanoma activity at low nanomolar concentrations.,We studied the effect of WP760 on several human melanoma cell lines derived from tumors in various development stages and having different genetic backgrounds.,WP760 inhibited cell proliferation (IC50 = 1-99 nM), impaired clonogenic cell survival (100 nM), and inhibited spheroid growth (≥300 nM).,WP760 did not induce double-stranded DNA breaks but strongly inhibited global transcription.,Moreover, WP760 caused nucleolar stress and led to activation of the p53 pathway.,PCR array analysis showed that WP760 suppressed transcription of ten genes (ABCC1, MTOR, IGF1R, EGFR, GRB2, PRKCA, PRKCE, HDAC4, TXNRD1, AKT1) associated with, inter alia, cytoprotective mechanisms initiated in cancer cells during chemotherapy.,Furthermore, WP760 downregulated IGF1R and upregulated PLK2 expression in most of the tested melanoma cell lines.,These results suggest that WP760 exerts anti-melanoma activity by targeting global transcription and activation of the p53 pathway and could become suitable as an effective therapeutic agent.,The online version of this article (doi:10.1007/s10637-017-0465-9) contains supplementary material, which is available to authorized users.