Edwin Kumah

Cutaneous squamous cell carcinoma (cSCC) leads to significant morbidity for patients with progression and metastases. However, the molecular underpinnings of these tumors are still poorly understood. Dissecting human cSCC pathogenesis amplifies the exigence for preclinical models that mimic invasion and nodal spread. This review discusses the currently available models, including two- and three-dimensional tissue cultures, syngeneic and transgenic mice, and cell line-derived and patient-derived xenografts. We further highlight studies that have utilized the different models, considering how they recapitulate specific hallmarks of cSCC. Finally, we discuss the advantages, limitations and future research directions.

Abstract One in thirteen organ transplant recipients (OTRs) develop post-transplant cutaneous squamous cell carcinoma (cSCC), leading to significant morbidity upon disease progression. Although chronic ultraviolet (UV) exposure primarily underlies keratinocyte carcinogenesis, long-term immunosuppression is a critical etiologic risk factor due to reduced systemic surveillance of dysplasia. Calcineurin inhibitors such as tacrolimus are commonly administered to suppress the immune system by blocking T-cell proliferation and preventing donor organ rejection. Contrarily, studies have evinced the off-target effects of immunosuppressives, namely azathioprine and cyclosporine, but not tacrolimus — the most widely used in all immunosuppression. In addition, the preclinical research literature has overlooked the contribution of stromal fibroblasts in the cSCC tumor microenvironment (TME). Hence, we hypothesize that tacrolimus exerts direct carcinogenic effects on keratinocytes and fibroblasts in the skin. We further postulate that tacrolimus enables a tumor-permissive microenvironment by activating dermal fibroblasts to cause aggressive and invasive keratinocyte carcinoma in OTRs. To examine this cellular crosstalk phenomenon, we used human keratinocytes, primary dermal fibroblasts and cSCC cells in mono- and co-cultures to mimic cutaneous and tumor complexities. Next, we assessed the molecular mechanisms utilized by tacrolimus to potentiate tumorigenesis pertinent to cell survival, proliferation and invasion. Herein, we show that tacrolimus promotes cell survival dose-dependently in cSCC cells. We also found that a fibroblast-cSCC spheroid co-culture significantly enhances cell invasion. In a wound-healing migration assay, tacrolimus increased the migratory capacity of normal keratinocytes and cSCC cells. However, cell proliferation remains nebulous due to cyclical dose-response effects in vitro. We then queried whether cell proliferation is affected in vivo using immunodeficient mice, and similar results were observed. We confirmed via immunoblotting that tacrolimus induced elevated expression of proliferation markers at distinct drug concentrations. Altogether, our findings illuminate keratinocyte-stroma interactions and particularly isolate the contributory role of tacrolimus in non-immune mediated cSCC pathogenesis. This knowledge suggests newer immunosuppressives that do not inhibit calcineurin activity may help relieve cutaneous malignancies in transplant recipients. Citation Format: Edwin Kumah, Yash Chhabra, Vania Wang, Agrani Dixit, Ashani Weeraratna, Kristin Bibee. Iatrogenic immunosuppression emplaces tacrolimus to drive squamous skin cancer progression and influence the microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 279.

Abstract Aging is an independent prognostic factor for melanoma, the most aggressive form of skin cancer. Although immune therapy has shown a dramatic improvement in melanoma treatment, resistance to it still occurs in some patients. Higher oxidative damage in tumor cells and lipid and iron accumulation are singular characteristics of the aged tumor microenvironment (TME). Here, we investigate the impact of these characteristics of the aged TME in promoting ferroptosis, a caspase-independent iron-mediated type of cell death, and as a possible therapeutic opportunity for older patients. Lipid peroxidation levels, resistance to ferroptosis inducers, and the impact of the aged TME secretome were assessed through in vitro and in vivo modeling. Initially, correlation analysis between transcriptomic data (TCGA-SKCM) and ferroptosis sensitivity (CTD^2) was performed to identify common denominators among transcripts associated with the aged TME and ferroptosis pathway. Phenotype-switching markers, such as AXL and Wnt5a, showed a strong correlation with several ferroptosis-related transcripts and an inverse correlation with ferroptosis resistance. Using a panel of melanoma cell lines with distinct Wnt5a levels, we confirmed this association and observed that three critical regulators of lipid peroxidation are downregulated in Wnt5a high lines: GPX4, AIFM2, and GCH1. The comparison through IHC of young and aged mouse skin samples highlighted a pro-ferroptosis signature increasing with age. Senescence markers such as p16 and p21 could be modulated in dermal fibroblasts through induction or inhibition of lipid peroxidation. When associating conditioned media (CM) derived from these dermal fibroblasts (capable of inducing phenotype switching), RSL-3, a GPX4 inhibitor, induced a higher lipid peroxidation and cell death in melanoma cells exposed to aged CM. To understand how ferroptosis impacts tumor growth in young and aged mice, we used a syngeneic tumor model injecting Yumm1.7 cells intra-dermally and the treatment with a lipid peroxidation inducer (imidazole ketone erastin, IKE) or an inhibitor (Liproxstatin-1). Tumors from aged mice showed stronger staining with 4-HNE antibody when compared to young, correlating with AXL and Wnt5a staining. Interestingly, both IKE and Liproxstatin-1 lead to age-specific reduced tumor growth, followed by changes in immune cell populations such as monocytes, macrophages and NK cells. We are currently exploring how these treatments impacted metastatic dissemination, stromal architecture and sensitivity in BRAF/MEK-resistant tumors. These results indicate that ferroptosis modulation is a promising avenue for combined treatment with existing clinical strategies. Citation Format: Murilo Ramos Rocha, Yash Chhabra, Alexis E. Carey, Cheyenne Palm, Kevin Zhang, Edwin Kumah, Vania Wang, Laura Huser, Elizabeth I. Harper, Fan Huang, Ashani Weeraratna. Ferroptosis modulates melanoma progression in an age-specific manner through changes in the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2558.

Abstract There is documented sex disparity in cutaneous melanoma incidence and mortality, increasing disproportionately with age and in the male sex. However, the underlying mechanisms remain unclear; while biological sex differences and inherent immune response variability have been assessed in tumor cells, the role of the microenvironment surrounding the tumor, contextually in aging, has been overlooked. We find that skin fibroblasts undergo age-mediated changes in their proliferation, senescence, ROS levels and stress response that vary with sex. We find that aged male fibroblasts selectively drive an invasive and slow-cycling phenotype in melanoma cells in vitro by increasing AXL expression. This is also evident in syngeneic mouse models where metastasis is increased in aged male mice. Mechanistically, intrinsic aging in male fibroblasts coupled with elevated ROS promotes EZH2 decline thereby increasing BMP2 secretion, which in turn drives the slower-cycling, highly invasive, and therapy-resistant melanoma cell phenotype, characteristic of the aged male TME. Inhibition of BMP2 activity blocks the emergence of the invasive phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. Our data provides an integrated view of how age and sex of the host contributes to melanoma progression and therapy responses. Bridging this knowledge gap will improve patient stratification and assist in tailoring the therapy to the individual. Citation Format: Yash Chhabra, Mitchell Fane, Sneha Pramod, Laura Hueser, Daniel Zabransky, Vania Wang, Edwin Kumah, Alexis Carey, Elizabeth Harper, Murilo Ramos Rocha, Ashani Weeraratna. Sexual differences in the aged melanoma tumor microenvironment dictates metastasis and therapeutic responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5513.