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The abnormality of ribosomal biogenesis is found to be involved in carcinogenesis and progression in various types of cancer. RPL22L1 gene is a highly homologous paralog of RPL22, both of which belong to the L22E family of ribosomal proteins and take part in encoding a cytoplasmic ribosomal protein that is a component of the 60S subunit of ribosomes. Our previous work has established a CRISPR/Cas9 genome-wide knockout screening profile and developed a two-component mixture model to identify cancer-specific essential genes in 436 cancer cell lines. Bioinformatics analysis reveals that RPL22L1 acts as a feature gene to predict cancer-specific essential genes in human cancer. Previous studies suggest that RPL22L1 is correlated with tumor progression and adverse prognosis in several cancer types, while its role in hepatocellular carcinoma (HCC) is unknown. In the present study, we found that RPL22L1 is highly expressed in embryonic stem cells (ES) and liver progenitor cells, which were acquired from a vitro hepatic differentiation model originating from ES cells. Hence, we hypothesized that RPL22L1 enhances cancer stem cell properties in HCC. Functional assays showed that RPL22L1 would promote sphere formation in vitro. qRT-PCR and Western Blotting results indicated that RPL22L1 could promote stemness-related gene expressions such as CD133, CD44, and SOX9 in HCC cell lines. Overexpression of RPL22L1 increased aldehyde dehydrogenase (ALDH) activity, which was decreased when RPL22L1 was silenced with short hairpin RNA (shRNA). Extreme limiting dilution assay showed that RPL22L1 overexpression stimulated self-renewal ability in immunodeficient mice. Furthermore, higher RPL22L1 expression can also promote cell proliferation and epithelial-mesenchymal-transition, as well as drug resistance to 5-Fluorouracil (5-FU) and cisplatin (CDDP). Silver nitrate staining demonstrated that RPL22L1 overexpression correlated with a larger size and number of nucleoli. The expression of RPL22L1 was also associated with adverse prognosis in HCC clinical samples. In conclusion, our study reveals aberrant ribosomal protein activity in HCC and the role of RPL22L1 in promoting HCC tumor progression. Citation Format: JinLin Huang, Zhenyang Guo, Jie Luo, Beilei Liu, Lanqi Gong, Xiaona Fang, Dora Lai-wan Kwong, Xinyuan Guan. Overexpression of ribosomal L22-like1 (RPL22L1) enhances cancer stem cell properties and promotes tumor progression in hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1489.

Background: Esophageal squamous cell carcinoma (ESCC) is the predominant form of esophageal cancer in developing areas and is among the top 10 deadliest cancers with a dismal 5-year survival rate of 10~20%. ESCC molecular pathogenesis has not been well characterized, while chemoradiotherapy remains the routine treatment option besides surgery, highlighting the need for a thorough understanding of ESCC and new treatment options. Tumor protein p63 (TP63), encoding p63, plays fundamental roles in stratified epithelial homeostasis. In the esophagus, ΔNp63 is the predominant isoform and is required for normal epithelial development. ESCC retains high ΔNp63 expression level, but its contribution to ESCC development is not fully understood. Results: We found ΔNp63 maintains ESCC development, depletion of which results in great attenuation and regression of cell line-derived xenograft growth in mice. Transcriptomic profiling showed type-I interferon (IFN-I) signaling-related pathways are the top pathways enriched in ΔNp63-depleted cells. This was further verified by quantitative PCR confirming up-regulations of interferon-stimulated genes upon ΔNp63 depletion in cell lines, and by transcriptomic profiling on our latest panel of naïve ESCC patient-derived organoids (PDOs) showing TP63 expression level negatively associated with IFN-I signaling-related pathways. Elevated endogenous retrotransposon-encoded RNA expression induces cancer cell IFN-I signaling through mediating tumor-suppressive viral mimicry response, an anti-viral state triggered by endogenous stimuli (1). We found cancer cell ΔNp63 depletion results in increased retrotransposon expression triggering dsRNA sensing and downstream signaling activation; Interferon regulatory factor 1 plays a critical role in mediating viral mimicry response downstream of ΔNp63. We further showed cells with lower ΔNp63 level and higher IFN-I signaling activity (ΔNp63-depleted cell lines and ΔNp63lo PDOs) display stronger responses to Decitabine, an anti-cancer drug and viral mimicry booster (1), as compared to control cell lines or ΔNp63hi PDOs, respectively. Conclusion: We identified a novel function of ΔNp63 in repressing cancer retrotransposon expression and explored the therapeutic potential of enhancing viral mimicry response, which may guide future ΔNp63/viral mimicry response-targeted therapy. Acknowledgement: We acknowledge the Research Grants Council (TRS T12-701/17-R to MLL) and the Food and Health Bureau (HMRF 06171566 to VZY) of Hong Kong SAR for funding supports. We acknowledge DSMZ for the KYSE cell lines. We acknowledge the HKUMed-CPOS for providing imaging facilities. Reference: 1.Chiappinelli KB, et al. Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses. Cell 2015 Citation Format: Valen Zhuoyou Yu, Shan Shan So, Bryan Chee-Chad Lung, Carissa Wing-Yan Wong, Ian Yu-Hong Wong, Claudia Lai-Yin Wong, Desmond Kwan-Kit Chan, Fion Siu-Yin Chan, Betty Tsz-Ting Law, Ka-On Lam, Anthony Wing-Ip Lo, Josephine Mum-Yee Ko, Wei Dai, Alfred King-Yin Lam, Dora Lai-Wan Kwong, Simon Law, Maria Li Lung. p63 constrains cancer cell transposable element expressions and viral mimicry response to sustain esophageal cancer development and indicates therapeutic vulnerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2611.

Advanced understanding of cancer immunology has contributed to a significant decline in cancer deaths in the past few decades. However, there are still vast majority of patients with no effective response for current immunotherapy drugs because of some innate immune cells, particularly macrophages and neutrophils, promoting all steps of malignant progression to metastasis. Therefore, the development of new strategies against these pro-metastatic non-tumor components in the tumor microenvironment is in need. Tumor-associated macrophage (TAMs), as the critical component of tumor microenvironment (TME), display diverse and even opposing function phenotypes, but without systematic investigation on their ubiquitous characteristics across different cancer types. Here, we perform pan-cancer analysis on 92 samples across 6 solid cancer types to profile the TME of normal, primary tumor, metastatic lymph node and distant metastasis at single-cell resolution, illustrating the commonalities/plasticity of heterogenous TAMs. We uncover 21 distinct subtypes of TAMs with varying abundance and biological function in different tissue microenvironment, providing evidence for the plasticity of TAMs. In particular, the enrichment of SPP1+MMP12+ macrophages in the TME of lymph node metastasis, likely interacting with metastasis-initiating cells (MICs), is associated with promoting malignant progression to metastasis and survival stratifications. Multi-color immunofluorescence (mIF) staining for relevant markers is being performed in mouse models and clinical samples for comprehensive validation. Our study comprehensively describes the characteristics and dynamics of TAMs and highlights their heterogeneity and plasticity depending on their surrounding microenvironment. Citation Format: Baifeng Zhang, Dora Lai-wan Kwong, Xin-Yuan Guan. Pan-cancer single-cell analysis reveals the interactions of SPP1+MMP12+ macrophages with metastasis-initiating cells that drives metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4699.

Background: Esophageal squamous cell carcinoma (ESCC) is one of deadliest esophageal malignancies in Asia. Cancer cachexia is a multifactorial body wasting syndrome and is one of the major causes of death in late-stage patients (1). It is characterized by skeletal muscle loss and atrophy, adipose and body wasting, and metabolic dysregulations and stress (1). ESCC is one of the cancer types with the highest morbidity and mortality rates due to cancer cachexia (2). However, there are few effective and approved treatments targeting cancer cachexia. Currently, cancer cachexia animal models are highly limited with few cancer types available as a pre-clinical drug testing platform, with none related to esophageal carcinoma (3). Results: Here we have developed a panel of ESCC patient-derived organoid xenograft (PDOX) mouse model based on our latest patient-derived organoid cultures originating from Hong Kong patients. Our immunodeficient mouse-based PDOX model resembles different cachexic phenotypes that cell line-derived xenograft lacks, including body (skeletal muscle and adipose tissue) wasting and functional signs (e.g. lack of body movement, reduced muscle strength, and loss of appetite). The slow-proliferating characteristic of the ESCC PDOX allows development of a cachexic condition before humane endpoint. Furthermore, our full panel of PDOX lines allowed us to identify the full spectrum, differential cachexia severity across mice bearing different PDOXs, indicating a heterogenous nature. We have comprehensively characterized the cachexia phenotypes and performed proof-of-concept preclinical drug tests using established anti-cachexic drugs; treated PDOX-bearing mice have improved body condition and function compared to the control, which were further verified by histological examinations of tissue samples. Molecular analysis showed that several classic cachexia related gene expressions were down-regulated in isolated liver (Il1b) and inguinal adipose tissues (adipose triglyceride lipase). Conclusions: Our ESCC PDOX model potentially serves as a great pre-clinical cachexia-targeted drug testing platform, providing more treatment options for ESCC patients for better disease management. Acknowledgement: We acknowledge the Research Grants Council (TRS T12-701/17-R to MLL) and the Food and Health Bureau (HMRF 06171566 to VZY) of Hong Kong Special Administrative Region for funding supports. We acknowledge DSMZ for the KYSE cell lines. We acknowledge the HKUMed-CPOS for providing imaging facilities. References:1. Baracos VE, et al. Cancer-associated cachexia. Nat Rev Dis Primers 2018;4:171052. Tanaka Y, et al. Recent advancements in esophageal cancer treatment in Japan. Ann Gastroenterol Surg 2018;2:253-653. Ballaro R, Costelli P, Penna F. Animal models for cancer cachexia. Curr Opin Support Palliat Care 2016;10:281-7 Citation Format: Bryan Chee-chad Lung, Valen Zhuoyou Yu, Ian Yu-hong Wong, Claudia Lai-yin Wong, Desmond Kwan-kit Chan, Fion Siu-yin Chan, Betty Tsz-ting Law, Ka-on Lam, Dora Lai-wan Kwong, Anthony Wing-ip Lo, Simon Law, Maira Li Lung. Modelling cancer cachexia using a comprehensive panel of heterogeneous esophageal squamous cell carcinoma xenografts for therapeutic application [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 363.

Background: Nasopharyngeal cancer (NPC) is associated with a poor survival rate. The ability of cancer cells to evade apoptosis and exhibit limitless replication potential allows for the progression of cancer. The aim of this study was to investigate in vitro the effect of the isoflavone phenoxodiol on human NPC cell line and T cells, which has previously been shown to inhibit cell proliferation and promote apoptosis in a range of cancer cell lines and in anti-CD3/anti-CD28-activated murine splenocytes. Methods: Four NPC cancer cell lines-C666, C17, and paired NPC43 EBV-positive and its respective EBV-negative cell lines were cultured in vitro, and then treated with phenoxodiol for 72h or 5 days. The migration and proliferation was investigated by transwell and XTT, respectively. Conditioned medium of treated NPC cell lines were collected and incubated with human PBMC for 5 days. CD4 and CD8 surface staining were analyzed by flow cytometry. Result: We demonstrated that phenoxodiol inhibited NPC migration and cell proliferation (IC50 4µM). Among all the NPC cell lines, the proliferation effect on EBV-negative cell line (NPC43-ve) is least responsive to phenoxodiol. A dose-dependent of apoptosis in NPC cell lines by phenoxodiol [early apoptotic cells (Annexin V+/PI- at 1µM); late apoptotic cells (Annexin V+/PI+ at 2µM), and necrotic cells (Annexin V-/PI+, at 4µM) were distinguished. Interestingly, phenoxodiol reduced frequency of CD4 T cells while promoting the number of CD8 T cells. Conclusions: Phenoxodiol demonstrates an ability in NPC cancer cells to induce its apoptosis while inhibit its migration and proliferation. Moreover, the ability of phenoxodiol to modulate T cells population indicates that phenoxodiol would be effective as a potential future treatment for NPC. Note: This abstract was not presented at the meeting. Citation Format: Ngar Woon Kam, See Wing Chan, George Sai-Wah Tsao, Xin-Yuan Guan, Dora Lai Wan Kwong. Nasopharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2957.