Single-cell RNA sequencing (scRNA-seq) has contributed to understanding cellular heterogeneity and immune profiling in cancer. The aim of the study was to investigate gene expression and immune profiling in colorectal cancer (CRC) using scRNA-seq. We analyzed single-cell gene expression and T cell receptor (TCR) sequences in 30 pairs of CRC and matched normal tissue. Intratumoral lymphocytes were measured with digital image analysis. CRC had more T cells, epithelial cells, and myeloid cells than normal colorectal tissue. CRCs with microsatellite instability had more abundant T cells than those without microsatellite instability. Immune cell compositions of CRC and normal colorectal tissue were inversely correlated. CD4 + or CD8 + proliferating T cells, CD4 + effector memory T cells, CD8 + naïve T cells, and regulatory T cells of CRC showed higher TCR clonal expansion. Tumor epithelial cells interacted with immune cells more strongly than normal. T cells, myeloid cells, and fibroblasts from CRCs of expanded T cell clonotypes showed increased expression of genes related to TNF and NFKB signaling and T cell activation. CRCs of expanded T cell clonotypes also showed stronger cellular interactions among immune cells, fibroblasts, and endothelial cells. Pro-inflammatory CXCL and TNF signaling were activated in CRCs of expanded T cell clonotype. In conclusion, scRNA-seq analysis revealed different immune cell compositions, differential gene expression, and diverse TCR clonotype dynamics in CRC. TCR clonality expansion is associated with immune activation through T cell signaling and chemokine signaling. Patients with CRCs of expanded clonotype can be promising candidates for immunotherapy.

BACKGROUND/AIM:Effective ex vivo maturation of dendritic cells (DCs) can increase the efficiency of cancer immunotherapy. We aimed to identify novel chemicals with the potential to differentiate and activate immature DCs (iDCs) to mature DCs (mDCs).MATERIALS AND METHODS:The expression of surface markers on THP-1 monocytes treated with the screened compounds was analyzed using FACS. Subsequent DC subset analysis and secreted cytokine profiling were also performed.RESULTS:FACS analysis showed that THP-1 cells treated with amsacrine hydrochloride, a DNA topoisomerase II inhibitor, exhibited the typical phenotype of conventional DCs (cDCs). The expression of DC activation markers was also increased after amsacrine treatment. The profile of cytokines produced by THP-1 cells treated with amsacrine was similar to that of mDCs.CONCLUSION:Amsacrine has an ex vivo capability of differentiating THP-1 monocytes into cDCs. As amsacrine has been used as a stable chemotherapeutic agent in humans, it can be useful for producing mDCs for cancer immunotherapy.

Background/Aims: The presence and clinical importance of tissue-resident memory T (TRM) cells have been recently described in association with various cancer types. However, the frequency and the traditional naïve–effector–memory phenotypic characteristics of TRM cells are largely unknown. Methods: We analyzed single-cell populations of colorectal cancer (CC, n = 18), stomach cancer (SC, n = 13), renal cell carcinoma (RCC, n = 19), and breast cancer (BC, n = 16) by dissociation of tumor tissue with collagenase/hyaluronidase. We investigated populations of naïve, effector, and memory T and TRM cells by flow cytometry. Results: Among CD8− cells, CC was associated with a significantly higher proportion of CD103+ T cells than other tumor types (p < 0.001). Among CD8+ cells, CC and SC were associated with higher CD103+ T-cell proportions than RCC and BC (p < 0.001). Significantly more CD8+ than CD8− cells expressed CD103 (p < 0.001). In association with SC, RCC, and BC, CD8+ T cells had a similar T-cell phenotype composition pattern: fewer effector T cells and more memory-type T cells among CD103+ cells compared with CD103− cells (p < 0.05). Tumors with higher proportion of CD103+ cells had no specific clinicopathologic characteristics than those with lower proportion of CD103+ cells. Conclusion: TRM cell abundance and phenotypes varied among CC, SC, RCC, and BC. Further studies regarding the functional differences of TRM associated with various tumors are warranted.

PURPOSE:The expression of major histocompatibility complex class I (MHC I) has previously been reported to be negatively associated with estrogen receptor (ER) expression. Furthermore, MHC I expression, level of tumor-infiltrating lymphocytes (TILs), and expression of interferon (IFN) mediator MxA are positively associated with one another in human breast cancers. This study aimed to investigate the mechanisms of association of MHC I with ER and IFN signaling. MATERIALS AND METHODS:The human leukocyte antigen (HLA)-ABC protein expression was analyzed in breast cancer cell lines. The expressions of HLA-A and MxA mRNAs were analyzed in MCF-7 cells in Gene Expression Omnibus (GEO) data. ER and HLA-ABC expressions, Ki-67 labeling index and TIL levels in tumor tissue were also analyzed in ER+/ human epidermal growth factor receptor 2 (HER2)- breast cancer patients who randomly received either neoadjuvant chemotherapy or estrogen modulator treatment followed by resection. RESULTS:HLA-ABC protein expression was decreased after β-estradiol treatment or hESR-GFP transfection and increased after fulvestrant or IFN-γ treatment in cell lines. In GEO data, HLA-A and MxA expression was increased after ESR1 shRNA transfection. In patients, ER Allred score was significantly lower and the HLA-ABC expression, TIL levels, and Ki-67 were significantly higher in the estrogen modulator treated group than the chemotherapy treated group. CONCLUSION:MHC I expression and TIL levels might be affected by ER pathway modulation and IFN treatment. Further studies elucidating the mechanism of MHC I regulation could suggest a way to boost TIL influx in cancer in a clinical setting.

A higher level of tumor-infiltrating lymphocytes (TILs) is associated with better prognosis in breast cancer patients. Adoptive transfer of lymphocytes coupled with conventional therapies has appealed to many clinicians and investigators as an effective treatment strategy for cancer patients, which necessitates efficient activation and expansion of cytotoxic T lymphocytes precisely targeting cancer cells. To comprehensively understand composition of TILs and to provide a grounding in adoptive T cell therapy, we analyzed the T cell receptor (TCR) repertoires in ex vivo–expanded TILs from nine breast cancer patients via next-generation sequencing. For the three of them, TCR repertoires of TILs gathered after the initial culture during 2 weeks were additionally analyzed and compared to those of TILs that underwent ex vivo rapid expansion procedure (REP). Diversity of TCR repertoire was variable among the patients. V/J segment usage in the clonotypes was similar among patients, with variable distribution of read counts for each V/J segment. The top 50% of most frequently observed VJ combinations was present in > 80% of the total clonotypes. Compared with TCGA data, the samples contained a similar amount of recurrent CDR3 sequences, but clonotype expansion was variable among the samples. In terms of clinicopathologic factor, presence of in vitro reactivity among triple-negative breast cancer cases seemed to be related to lower Shannon’s index, but p value was not statistically significant. In addition, the proportion of CD45RO+ cells out of CD8+ T cells were negatively correlated with Shannon’s diversity index for both TCRα and TCRβ chains (p = 0.010) via Spearman test. In this study, we identified a heterogeneous pattern of expanded T cell clones and stable usage of V/J segments in ex vivo–expanded TILs from breast cancer patients. Further large-scale studies are requisite to elucidate the clinical significance of TCR repertoires.

BACKGROUND:The clinical significance of adoptive tumor-infiltrating lymphocyte (TIL) therapy has been demonstrated in many clinical trials. We analyzed the in vitro reactivity of cultured TILs against autologous breast cancer cells.METHODS:TILs and cancer cells were cultured from 31 breast tumor tissues. Reactivity of TILs against cancer cells was determined by measuring secreted interferon-gamma. Expression levels of epithelial markers, major histocompatibility complex molecules, and programmed death-ligand 1 (PD-L1) in cancer cells, and T cell markers (memory, T cell activation and exhaustion, and regulatory T cell markers) in expanded TILs were analyzed and compared between the reactive and non-reactive groups.RESULTS:In seven cases, TILs showed reactivity to autologous cancer cells. Six of these cases were associated with triple-negative breast cancer (TNBC). All reactive TNBCs were derived from surgical specimens after neoadjuvant chemotherapy (NAC). Higher expression of Ki67 in tumor tissues and lower expression of PD-L1 in cultured cancer cells were associated with reactivity. Proliferation of reactive TILs was high. High proportions of T cells and PD-1+CD4+ and PD1+CD8+ T cells were associated with reactivity in TNBC cases, while other activation or exhaustion markers were not.CONCLUSION:TILs from approximately half the TNBC cases with NAC showed reactivity against autologous cancer cells. The proportion of PD-1+ T cells was higher in the reactive group. Adoptive TIL therapy combined with PD-1 inhibitors might be promising for TNBC patients with residual tumors after NAC.

Abstract Background: Clinical significance of tumor-infiltrating lymphocytes (TILs) was demonstrated in many tumor types. In the clinical trials, immune checkpoint inhibitors showed significant clinical efficacy in some tumor types with high mutation burden such as melanoma, bladder cancer, and smoking-induced lung cancer, while there were little effects in cancers with lower mutation rates such as breast cancer. Recently, selection and adoptive transfer of autologous mutation specific TILs showed successful regression of metastatic cancer in an estrogen receptor-positive breast cancer patient. In this study, we analyzed in vitro reactivity of cultured TILs against autologous breast cancer cells. Methods: We cultured TILs and cancer cells derived from 31 breast tumor tissues obtained from the surgery (13 of hormone receptor-positive, one of HER2-positive, and 17 of triple-negative breast cancer [TNBC]). Reactivity of TILs against cancer cells was determined by IFN-gamma ELISA analysis after co-culture of TILs and cancer cells for 24 hours. Epithelial cell markers (cytokeratin and EpCAM) for cancer cells and T cell markers [memory types (CD45RO, CCR7, and CD62L), T-cell activation and exhaustion markers (PD-1, 4-1BB, OX40, CD39, CD107a, TIM3, TIGIT, KLRG1, and LAG3), and regulatory T cells markers (CD25, CD127, and FOXP3)] for expanded TILs were analyzed by flow cytometry. Results: Seven cases showed in vitro reactivity, which was defined by increased secretion (more than twice) of IFN-gamma from TILs upon cancer cell interaction. Interestingly, most of reactive TILs were from TNBC (6/7) and all of TNBCs showing in vitro reactivity were derived from resection specimens after neoadjuvant chemotherapy (NAC, 6/11). The remaining one case with in vitro reactivity was derived from HER2-positive breast cancer. TNBC cancer cells of cases with in vitro reactivity had more EpCAM-positive cells (p = 0.02) than those of nonreactive cases. Most T cells in TIL cultures showed effector memory phenotype (CD45RO+/CCR7-; CD4 T cells, median 97.4 % (73.7-99.9); CD8 T cells, median 98.8% (93.8-99.8)). TILs of TNBC cases with in vitro reactivity expressed higher levels of PD-1 (p value < 0.01) on CD4 T cells. PD-1 expression levels on CD8 T cells in reactive group tended to be higher than nonreactive group (p = 0.05). Expression of other markers was not different between reactive and nonreactive groups. Conclusion: Among 17 TNBC cases, 35% showed in vitro TIL reactivity against autologous tumor cells. All reactive TNBCs were NAC-resistant tumors, which have high risk of tumor recurrence and high mortality rates. Adoptive TIL therapy might be a promising therapeutic modality for TNBC patients. Citation Format: Heejae Lee, Young-Ae Kim, Hye Seon Park, Won Seon Bang, In Ah Park, Miseon Lee, Gyungyub Gong, Hee Jin Lee. Antitumor reactivity of human breast tumor-infiltrating lymphocytes (TILs) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A41.

Background: Tumor-infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLSs) are prognostic markers in triple-negative breast cancer (TNBC). Our study analyzed the relationship between cluster of differentiation (CD)11c-positive dendritic cells (DCs) and TILs and TLSs to elucidate mechanisms of TIL influx. Materials and Methods: Immunohistochemical staining for CD4, CD8, and CD11c in tissue microarrays from 681 patients with TNBC was performed. The proportions of TILs and TLSs were reviewed. Two additional TNBC gene expression datasets were used. Results: CD11c expression showed a significantly positive correlation with the level of TILs and the number of CD4+ and CD8+ T-cells, as well as an abundance of TLSs. CD11C gene expression was also significantly correlated with expression of CD4, CD8, and genes related to TLSs in both datasets. Conclusion: We demonstrated a strong correlation of CD11c expression, which represents DCs, with TILs and TLSs in TNBC. Further investigation is warranted to identify therapeutic modalities that facilitate recruitment and activation of DCs.

Abstract Background: The presence and clinical importance of tissue-resident memory T cells (TRM) have been recently described in human lung, liver, and bladder cancer. However, the frequency and phenotypic characteristics of TRM in other tumors are largely unknown. Methods: We analyzed single-cell populations of renal cell carcinoma (n=20), breast (n=16), colorectal (n=20), and stomach cancer (n=15) by dissociation of tumor tissue with collagenase/hyaluronidase. We investigated population of memory T cells and TRM using anti-CD45RO or anti-CD103 antibodies by flow cytometry. Results: Colorectal cancer had higher level of CD4+ T cells (70.1%) among CD3+ T cells than other tumors (stomach cancer, 59.6%; renal cell carcinoma, 49.4%; breast cancer, 49.0%; p=0.001). In CD4+ T cells, the percentage of CD45RO+ memory T cells was higher in renal cell carcinoma (75.7%) and breast cancer (85.4%) than colorectal (36.9%) and stomach cancer (38.1%, p<0.001). About 4% of CD4+ T cells in renal cell carcinoma, colorectal and stomach cancer were TRM, while 14.3% of CD4+ T cells were TRM in breast cancer (p<0.001). The percentage of CD45RO+ memory T cells among CD4+ TRM was about 60% in all tumors (p=0.164). In CD8+ T cells, the percentage of CD45RO+ memory T cells was higher in renal cell carcinoma (73.5%), breast (66.4%) and stomach cancer (54.4%) than colorectal cancer (33.0%, p<0.001). Stomach cancer had higher level of TRM (74.3%) among CD8+ T cells than other tumors (colorectal cancer, 19.6%; renal cell carcinoma, 20.1%; breast cancer, 30.6%; p<0.001). The percentage of CD45RO+ TRM in renal cell carcinoma (89.3%) and breast cancer (89.5%) was higher than colorectal (43.0%) and stomach cancer (51.5%, p<0.001). Conclusions: The frequency and phenotypic characteristics of TRM in renal cell carcinoma, colorectal, stomach, and breast cancer were different. Further studies regarding functional difference and clinical significance of TRM in various tumors are warranted.Acknowledgement: This study was supported by Basic Science Research Programs through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning, Republic of Korea (NRF-2017R1D1A1B03033104). Citation Format: Hye Seon Park, Young-Ae Kim, Won Seon Bang, Heejae Lee, Miseon Lee, In Ah Park, In Hye Song, Sun-Hee Heo, Hyeonjin Lee, Hee Jin Lee, Gyungyub Gong. Phenotypic difference of CD103+ tissue-resident memory T cells in various cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4685.

Abstract Background: The level of tumor-infiltrating lymphocytes (TILs) and the presence of tertiary lymphoid structures (TLSs) are significant prognostic and predictive factors in primary breast cancer. However, understanding about the differences of TILs and TLSs among different metastatic sites or between primary breast tumors and metastatic sites is limited. Methods: A total of 337 cases of metastatic breast cancer with available hematoxylin and eosin slides of metastatic sites (biopsy, n=185; operation, n=152) were included. We analyzed the percentage of TILs (defined as the percentage of invasive carcinoma stroma infiltrated by lymphocytes in 10% increments; if less than 10% of stroma was infiltrated by TILs, 1% or 5% criteria were used; all available full sections were evaluated) and the presence of TLSs (defined as the lymphoid aggregation with high endothelial venules) in primary and metastatic sites. Results: The mean percentage of TILs in the lung (19.5%) was significantly higher than those in the liver (4.2%), brain (8.3%), and ovary (3.4%, p<0.001). The level of TILs in metastatic tumors of the lung was higher than those in primary tumors (Wilcoxon signed-rank test, p<0.001), while metastatic tumors of the liver (p=0.024) and brain (p=0.095) showed lower level of TILs than in primary tumors. TLSs were found only in the lung (27%) and liver (13%). Patients with lung metastasis (p<0.001), higher level of TILs (p=0.001), and presence of TLS (p=0.002) showed better overall survival. In patients with lung metastasis, who showed best clinical outcome, those with 10% or more of TIL had better overall survival than those with less than 10% (p=0.008). Conclusion:. Metastatic breast tumors in the lung had more TILs than other sites and matched primary tumors. Underlying mechanism of differences of TILs among metastatic sites should be investigated. Citation Format: Miseon Lee, Hee Jin Lee, In Hye Song, In Ah Park, Sun-Hee Heo, Young-Ae Kim, Won Seon Bang, AReum Lee, Hyeonjin Lee, Heejae Lee, Gyungyub Gong. Comparison of tumor-infiltrating lymphocytes between primary and metastatic tumors in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 697.

The level of tumor-infiltrating lymphocytes and presence of tertiary lymphoid structures are significant prognostic and predictive factors in primary breast cancer. However, the understanding about differences in tumor-infiltrating lymphocytes and tertiary lymphoid structures at various metastatic sites or between primary breast tumors and metastatic sites is limited. A total of 335 cases of metastatic breast cancer from four metastatic sites (lung, liver, brain, and ovary) were included. We analyzed the percentages of tumor-infiltrating lymphocytes and presence of tertiary lymphoid structures in the primary and metastatic sites. The mean level of tumor-infiltrating lymphocytes in the lung metastases was higher than in the liver, brain, ovary, and matched primary tumors, while metastatic tumors of the liver and brain showed lower levels of tumor-infiltrating lymphocytes than primary tumors. Tertiary lymphoid structures were only found in the lung and liver, and in cases of brain metastases the change of tertiary lymphoid structures from present to absent significantly affected the level of tumor-infiltrating lymphocytes in metastases compared with that in matched primary tumors. Patients with a lower histological grade, hormone receptor positivity in primary tumors and metastases, a lower level of tumor-infiltrating lymphocytes and absence of tertiary lymphoid structures in primary tumors, a higher level of tumor-infiltrating lymphocytes and presence of tertiary lymphoid structures in metastases, and lung metastases showed significantly better overall survival. Our results showed that metastatic breast tumors in the lung had more tumor-infiltrating lymphocytes than did tumors at other sites and matched primary tumors. In addition, the presence of tertiary lymphoid structures in metastatic sites is a critical factor for the level of tumor-infiltrating lymphocytes.

Ginsenosides are the principal active components of ginseng and are considered attractive candidates for combination cancer therapy because they can kill tumors and have favorable safety profiles. However, the overall benefit of ginsenosides remains unclear, particularly in cancer immunosurveillance, considering the controversial results showing repression or promotion of immune responses. Here we identify a potentiating role of ginsenoside F1 (G-F1) in cancer surveillance by natural killer (NK) cells. Among 15 different ginsenosides, G-F1 most potently enhanced NK cell cytotoxicity in response to diverse activating receptors and cancer cells. G-F1 also improved cancer surveillance in mouse models of lymphoma clearance and metastatic melanoma that rely on NK cell activity. G-F1-treated NK cells exhibited elevated cytotoxic potential such as upregulation of cytotoxic mediators and of activation signals upon stimulation. NK cell potentiation by G-F1 was antagonized by insulin-like growth factor (IGF)-1 blockade and recapitulated by IGF-1 treatment, suggesting the involvement of IGF-1. Thus, our results suggest that G-F1 enhances NK cell function and may have chemotherapeutic potential in NK cell-based immunotherapy. We anticipate our results to be a starting point for further comprehensive studies of ginsenosides in the immune cells mediating cancer surveillance and the development of putative therapeutics.

Activation of NK cells is triggered by combined signals from multiple activating receptors that belong to different families. Several NK cell activating receptors have been identified, but their role in the regulation of effector functions is primarily understood in the context of their individual engagement. Therefore, little is known about the signaling pathways broadly implicated by the multiple NK cell activation cues. Here we provide evidence pointing to glycogen synthase kinase (GSK)-3β as a negative regulator of multiple NK cell activating signals. Using an activation model that combines NKG2D and 2B4 and tests different signaling molecules, we found that GSK-3 undergoes inhibitory phosphorylation at regulatory serine residues by the engagement of NKG2D and 2B4, either individually or in combination. The extent of such phosphorylation was closely correlated with the degree of NK cell activation. NK cell functions, such as cytokine production and cytotoxicity, were consistently enhanced by the knockdown of GSK-3β or its inhibition with different pharmacological inhibitors, whereas inhibition of the GSK-3α isoform had no effect. In addition, NK cell function was augmented by the overexpression of a catalytically inactive form of GSK-3β. Importantly, the regulation of NK cell function by GSK-3β was common to diverse activating receptors that signal through both ITAM and non-ITAM pathways. Thus, our results suggest that GSK-3β negatively regulates NK cell activation and that modulation of GSK-3β function could be used to enhance NK cell activation.