Background: Tenascin-C (TNC) is an extracellular matrix protein that is widely expressed in the stromal fibroblasts of various cancers. However, the roles of TNC in colorectal cancer (CRC) cells remain unclear. Methods: The expression of TNC, cancer stem cell-like (CSC) and cell cycle markers, and Hedgehog (HH) signaling pathway genes were assessed in 100 paraffin embedded clinical CRC patient tissues using immunohistochemistry. The interaction between TNC and CSC marker or HH related genes in CRC cells were detected by immunofluorescence. Cell cycle distribution was measured by flow cytometry. Migration and invasion were evaluated by transwell assays. The expressions of TNC, CSC marker, and HH related proteins were analyzed by western blot. Results: TNC expression was markedly upregulated in CRC tissues, and was associated with worse clinical outcomes. TNC overexpression was positively associated with CSC marker LSD1, cell cycle markers CDK4 and p16, and HH signaling pathway related genes SMO and GLI1 in clinical CRC tissue samples. TNC silencing downregulated the expression of the CSC marker LSD1, and the proliferation, migration, and invasion of CRC cells. Interestingly, the GLI1 inhibitor GANT61 strongly inhibited the expression of TNC in CRC cells. Conclusions: TNC may drive tumor progression and is involved in CSC properties via the HH signaling pathway. TNC has potential value in the evaluation of poor prognosis in CRC.

To understand the potential effects of cancer cells on surrounding normal mammary epithelial cells, we performed direct co-culture of non-tumorigenic mammary epithelial MCF10A cells and various breast cancer cells. Firstly, we observed dynamic cell–cell interactions between the MCF10A cells and breast cancer cells including lamellipodia or nanotube-like contacts and transfer of extracellular vesicles. Co-cultured MCF10A cells exhibited features of epithelial-mesenchymal transition, and showed increased capacity of cell proliferation, migration, colony formation, and 3-dimensional sphere formation. Direct co-culture showed most distinct phenotype changes in MCF10A cells followed by conditioned media treatment and indirect co-culture. Transcriptome analysis and phosphor-protein array suggested that several cancer-related pathways are significantly dysregulated in MCF10A cells after the direct co-culture with breast cancer cells. S100A8 and S100A9 showed distinct up-regulation in the co-cultured MCF10A cells and their microenvironmental upregulation was also observed in the orthotropic xenograft of syngeneic mouse mammary tumors. When S100A8/A9 overexpression was induced in MCF10A cells, the cells showed phenotypic features of directly co-cultured MCF10A cells in terms of in vitro cell behaviors and signaling activities suggesting a S100A8/A9-mediated transition program in non-tumorigenic epithelial cells. This study suggests the possibility of dynamic cell–cell interactions between non-tumorigenic mammary epithelial cells and breast cancer cells that could lead to a substantial transition in molecular and functional characteristics of mammary epithelial cells.

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As a new promising in vivo platform for personalized treatment in the field of cancer research, the most improved way has been adopted by far is grafting patient-derived xenograft (PDX) to humanized mouse models which represent the unique features of interaction with human immune system and tumor microenvironment (TME). Here, we engineered triple negative breast cancer (TNBC) humanized PDX mouse (Hu-PDX) model to observe human immune system and tumor growth in TME. In addition, we analyzed gene ontology of PDX isolated from hu or non-humanized mouse model. Method: Human CD34+hematopoietic stem cells (HSCs) derived from cord blood is reconstructed into mature human leukocytes (hCD45+) in NOD.Cg-PrkdcscidIL2γgtm1 Sug (NOG) mice. TNBC PDX tumors are established it with unmatched human leukocyte antigen (HLA) genotypes, confirmed by that hCD45+ engraftment levels did not exceed 20-30% in the peripheral blood of mice. To be repopulated functional human T cells in xenograft models with severely depleted thymus, hCD34-PBMC cells are directly intravenously injected into tail vein of humanized mice followed by hCD34+cells injected without the onset of graft-versus-host disease (GVHD). The growth and gene expression of TNBC PDX tumors, as well as engrafted human immune population including T, B, natural killer (NK) cells and myeloid cells in TME, are monitored and investigated in both hu and non-hu-mice model. Results: Human immune reconstruction of cells, of the lymphoid as well as myeloid lineages, has been successfully accomplished in peripheral blood, spleen, and bone marrow over time in TME. Although the number of human mature leukocytes was gradually decreased in mice by 17 weeks, humanized mice have enabled studies of human immunity and TME in limitation period. Also, one of remaining limitations is unable to positive and negative selection of human T cells in the humanized mouse thymus and required to human major histocompatibility complex (MHC) molecules in humanized mice. To overcome this barrier, additional stimulation factor for generating functional T cells have successfully engrafted by transferring hCD34-PBMC cells. Moreover, tumor infiltrating human lymphocytes (TILs) have obviously detected in Hu-PDX model, although the differences tumor growth rate was not significantly monitored, compared with non-humanized mice. Interestingly, the analysis of gene ontology; biological process, cellular component and molecular function, ribosomal relevant proteins, not immune related genes, were highly up-regulated in TNBC PDX humanized in comparison to non-humanized mouse model. Conclusion: Humanized PDX model will contribute to studies more efficiently for the machinery underlying immunological response in breast tumor microenvironment. Furthermore, it might be an important unknown mechanism that the effect of elevated cancer-related ribosomal proteins expression within hu immune-TME. Citation Format: Yujeong Her, Hye Yeon Son, Ju Hee Kim, Woo Hang Heo, Mingji Quan, Songbin Li, Hyeong Gon Moon. Development of humanized patient-derived xenograft models for triple negative breast 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 6258.

Objective Immunotherapy using the tumor-specific antigens (TSAs) is a promising strategy in breast cancer. Studies have suggested that the in vivo exposures to certain tumors can induce adaptive anti-tumor immunity in syngeneic tumor models. In this study, we show the efficacy of the tumor lysate vaccine and peptide-based vaccine against tumor neoantigen in suppressing tumor growth and metastasis in 4T1 syngeneic tumor models. Method We used BALB/c mice and its syngeneic tumor cell lines to evaluate the anti-tumor effect induced by the transient exposure to the tumor cells. For tumor vaccines, we synthesized the tumor lysate vaccine by the freeze-thaw method or synthetic peptide against the selected tumor neoantigens identified by exome sequencing. We systemic and local immune remodeling was investigated by using immunohistochemistry, flow cytometry, and single cell RNA sequencing. Results We observed a significant reduction of tumor growth and metastasis in 4T1 syngeneic tumors when the mice were previously exposed to the same cells (pre-exposure group). This anti-tumor effect induced by the exposures to the tumor was cell line-specific. The 4T1 tumor lysate vaccines administered prior to the tumor cell injection also showed significant inhibitory effect on tumor growth and metastasis. T lymphocytes, isolated from the tumor tissues of the 4T1 pre-exposure mice and lysate vaccine-treated mice, showed higher levels of TNF-ɑ and IFN-ɣ when compared to the control those from the control tumors. The lysate vaccine treatment resulted in a substantial remodeling of tumor microenvironment including reduction of myeloid-derived suppressor cells and M2 tumor-associated macrophages. On the other hand, the numbers of M1 tumor-associated macrophages and effector memory CD8+ T cells were increased by the lysate vaccine. While the peptide vaccine showed no inhibitory effect on the primary tumor growth, it also suppressed spontaneous lung metastasis. Finally, we administered lysate tumor vaccine after the tumor establishment to determine the therapeutic effect. The lysate vaccine significantly suppressed the tumor growth and lung metastasis of the syngeneic 4T1 tumors. Conclusion Tumor lysate vaccine can suppress the tumor growth and metastasis in the 4T1 syngeneic mouse models by inducing substantial remodeling of tumor immune microenvironment. Additionally, tumor lysate vaccine can elicit similar anti-tumor immune response when administered after the establishment of the primary tumor suggesting a potential therapeutic value. Citation Format: Hyeong-Gon Moon, Hye Youn Son, Woo Hang Heo, Mingji Quan, SONGBIN LI, Haritonova Valentina, Hamin Jeong, Wonshik Han, Han-Byoel Lee, YUJEONG HER, Ju Hee Kim. Personalized tumor vaccine can suppress tumor growth and metastasis in the syngeneic mouse breast cancer model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-05.