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Supplemental Table 1, NMR Results, Supplemental Table 2, Supplemental Figures 1 and 2

Urban areas provide a range of benefits to sustain human livelihood and contribute to human well-being through urban ecosystem services. Open spaces in core urban areas of Kathmandu valley in Nepal carries multiple advantages of stimulating social cohesion, offers safe area immediately after a crisis induced by natural hazards, contributes in environmental improvement and mitigates urban flooding. In most urban areas of Nepal, unplanned urbanization has resulted in alteration of landscapes from permeable vegetated surfaces to a series of impervious interconnected surfaces resulting in large quantities of stormwater runoff, requiring wider implementation of water sensitive urban design. After 2015 Gorkha earthquake, several blue-green infrastructure projects are implemented by local governments inside Kathmandu valley in open spaces. This paper presents application examples of green infrastructure projects and through case studies provides a framework for optimization of green infrastructure systems in Nepal. The paper also provides a practitioners perspective on the current state of knowledge, highlights technical challenges in green infrastructure implementation in Kathmandu and points out recommendations to overcome them.

Colorectal cancer (CRC) is a highly complex disease with multiple risk factors. The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in several cancers and is a negative prognostic factor for cancer patient survival. Previous reports indicate a potential role for overexpression of NR4A1 in T-cell exhaustion and in this study, we aim to investigate the antitumorigenic activity of two bis-indole derived ligands (DIMs) that act as receptor antagonists. Immune competent C57BL/6 mice and mouse MC-38 colon cancer cells were used and tumor Infiltrating Lymphocytes (TILs) were isolated from mice either untreated or treated with CDIM/NR4A1 antagonists. FACS analysis and Real -Time PCR were performed to determine expression of exhaustion markers in these tumor T-cell population. 1,1-Bis(3΄-indolyl)-1-(3-bromo-5-trifluoromethoxyphenyl)methane (DIM-3-Br-5-OCF3) and the 3,5-dichlorophenyl analog (DIM-3,5-Cl2) at doses of 2.5 and 7.5 mg/kg/d inhibited tumor growth and downregulated expression of PD-L1, an NR4A1-regulated gene in MC-38 - derived tumors and cells. The mouse MC-38 colon cancer cell line was used in a syngeneic mouse model of colon cancer and tumor infiltrating lymphocytes (TILs) from MC-38 cell-derived colon tumors exhibited multiple markers of T-cell exhaustion in both CD8+ and CD4+ T-cells. Lymphocytes from an enlarged spleen in these tumor-bearing animals also exhibited markers of CD8+ and CD4+ T-cell exhaustion. Analysis of CD8+ cells from TILs showed that treatment with the NR4A1 antagonists modulated expression of several genes associated with T-cell exhaustion namely a decrease in TOX, TOX2 and NFAT mRNAs, activation of T-Bet. The percentage of CD8+ and CD4+ T-cells from tumors and spleen expressing PD-1, 2B4 and TIGIT was decreased in the treated vs control mice and TIM3 expression was also decreased in CD8+ (tumors and spleen) and CD4+ (tumors) T-cells. These findings suggest that NR4A1 antagonists are highly effective as anticancer agents in this mouse syngeneic colon tumor model by inactivating the pro-oncogenic activities of NR4A1 in tumors and by remediating NR4A1-regulated T-cell exhaustion in tumor and splenic lymphocytes. Citation Format: Kumaravel Mohankumar, Gus Wright, Subhashree Kumaravel, Rupesh Shrestha, Lei Zhang, Maen Abdelrahim, Robert S. Chapkin, Stephen Safe. Nuclear receptor 4A1 ligands target T-cell exhaustion in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 236.