Breast Cancer Microenvironment Research Articles

Integrative single-cell sequencing analysis distinguishes survival-associated cells from the breast cancer microenvironment.

Breast cancer shows a highly complex tumor microenvironment by containing various cell types. Identifying prognostic cell populations in the tumor microenvironment will improve the mechanistical understanding of breast cancer and facilitate the development of new breast cancer therapies by targeting the tumor microenvironment. The development of single-cell sequencing reveals various cell types, states, and lineages within the context of heterogenous breast tumors, but identifying phenotype-associated subpopulations is challenging. Here, we applied Scissor (single-cell identification of subpopulations with bulk Sample phenotype correlation) to integrate single cell and bulk data of breast cancer, and found that MHC-deficient tumor cells, FABP5+ macrophages, and COL1A1+ cancer-associated fibroblasts (CAFs) were detrimental to patient survival, while T cells and dendritic cells were the main protective cells. MHC-deficient tumor cells show strong downregulation of MHC expression for immune evasion by downregulating interferon and JAK-STATs signaling. FABP5+ macrophages show low antigen-presenting activity via associating with lipid metabolism. Our data suggest that COL1A1+ CAFs may block T-cell immune infiltration through cell interaction in breast tumor microenvironment. Taken together, our study reveals survival-associated subpopulations in breast tumor microenvironment. Importantly, subpopulations related to immune evasion of breast cancer is uncovered.

Abstract 5087: Combination therapy of immune checkpoint inhibitor and HIF1α/c-MET peptide vaccine suppresses metastasis by modulating tumor microenvironment in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 20% of all breast cancer and has limited therapeutic option except for immunotherapy because ER, PR, and HER2 targeted for treatment are absent. It also tends to be more aggressive meaning more likely to metastasize faster to other organs such as lungs, liver, and bone, and its recurrence rate is high which leads to a poor prognosis. Immunotherapy is an option to treat TNBC such as programmed death ligand 1 (PD-L1) antibody. However, tumor cells use various immune evasion mechanisms in addition to PD-1/PD-L1 pathway to interrupt the effect of immunotherapy. Moreover, tumor microenvironment (TME) is one of the important factors for the effective anti-tumor immune responses from immunotherapy. Hypoxia is a typical event in TNBC. Hypoxia inducible factor 1-alpha (HIF1α) is a transcription factor that regulates angiogenesis by expressing VEGF, and in addition, upregulates the c-MET which increases the proliferation, migration, and survival of tumor cells. Moreover, HIF1α directly regulates the expression of CD47. CD47 is a transmembrane protein that binds to SIRPα of macrophages to inhibit the phagocytosis of macrophages. Therefore, the efficacy of immune checkpoint inhibitors decreases even if there is a higher degree of tumor-infiltrating lymphocytes (TILs). Combination therapy with immune checkpoint and tumor associated antigens (TAAs) derived peptide vaccine could inhibit metastasis. In present study, we found that HIF1α/c-MET peptide vaccination delayed metastasis in C3(1)Tag mouse. We also investigated whether combination therapy with immune checkpoint inhibitors and peptide vaccine inhibited metastasis. Next, we observed that combination strategy of HIF1α/c-MET peptide vaccine and treated anti-PD-L1 antibody or/and anti-CD47 antibody was more effective in systemically inoculated M6 cells growth in C3(1)Tag mice. Immunohistochemical analysis of the tumors showed that the protein expression of CD47, HIF1α, and c-MET were significantly decreased. On the other hand, CD4+ T cells, CD8+ T cells and M1 macrophages increased, but M2 macrophages decreased, resulting in delayed metastasis in combination therapy group compared to single therapy and control group. In addition, IFNγ ELISPOT showed that peptide vaccination significantly increased the HIF1α/c-MET specific IFNγ-secreting T cell response in splenocytes. We also assessed osteoclastogenesis in bones, which showed reduction of osteoclast in combination therapy group compared to single therapy and control group.Taken together, combination therapy with immune checkpoint inhibitor targeting PD-L1 and CD47 and peptide vaccine appears to be a promising strategy that inhibit metastasis by modulating tumor microenvironment in triple negative breast cancer. Citation Format: JinHwa Hong, Jimin Lee, Soon Young Lim, Ju Won Kim, Ah Reum Lim, Kyong Hwa Park. Combination therapy of immune checkpoint inhibitor and HIF1α/c-MET peptide vaccine suppresses metastasis by modulating tumor microenvironment in triple-negative breast cancer. [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 5087.

Abstract 2506: The influence of sex on the immune microenvironment in male and female breast cancer

Abstract Even when accounting for multiple clinical parameters, male breast cancer patients have significantly lower survival rates than females. Males and females differ in their levels of sex hormones androgens and estrogens. These sex hormones have varying influences on immune cell populations, which in turn may affect disease progression. Thus, the sex of the patient and the levels of sex hormones are likely important for anticancer immunity. Here, we assessed the importance of sex hormone signaling in the immune cells (host) versus the tumor cells for defining the immune landscape associated with primary and metastatic tumors. We used flow cytometry to determine the immune profile of mammary tumors, spleens, and lungs, and histochemistry to evaluate metastasis in transgenic MMTV-PyMT and in mice orthotopically injected with sex-specific MMTV-PyMT-derived cell lines in male and female cohorts. In transgenic mice, males had greater proportions of CD4+ T-helper and total myeloid cells in all organs analyzed. The splenic and pulmonary immune profiles of tumor-bearing males were more similar to cancer-free littermates than in females. Interestingly, males trended towards increased metastatic colonization in the lungs. Sex-associated immune profiles differed between transgenic and orthotopic mice. In mice bearing male-derived R221a PyMT tumors, females had decreased intratumoral neutrophils and elevated natural killer cells with increased splenic B-cells in comparison to males. Overall, our data supports a sex-dependent immune response in mammary carcinoma that is, in part, regulated by the hormonal environment of the host. With emerging therapeutics targeting the tumor immune microenvironment, characterization of immune profiles is critical for optimizing their use in all breast cancer patients. Citation Format: Ebony Hargrove-Wiley, Barbara Fingleton. The influence of sex on the immune microenvironment in male and female breast cancer [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 2506.

Abstract 2507: Chronic stress promotes tumor progression and metastasis through the tumor microenvironment in triple-negative breast cancer

Abstract Background: Breast cancer is the most common cancer diagnosed in American women. Triple-negative breast cancer (TNBC) is three times more likely to end in death than other breast cancer subtypes and disproportionately affects young African American women. Chronic stress as a risk factor promotes tumor progression, but its role in TNBC progression and metastasis remains elusive. Methods: In this study, mice were implanted with 4T1-luciferase cells in the tibia or the fourth mammary pad of BALB/c mice and NSG mice. After four weeks of restraining inducing chronic stress and (or) β-adrenergic receptor inhibitor treatment, the impact of chronic stress on the progression and metastasis of tumor, tumor microenvironment was assessed. Results: Our results showed that chronic stress facilitated tumor progression and metastasis in both BALB/c mice and NSG TNBC mice models. In contrast, chronic stress reduced the number and function of tumor-infiltrating TCD8+ cells but increased the expression of the β2-adrenergic receptor of TCD8+ cells in the TNBC tumor microenvironment (TME) of BALB/c mice. Interestingly, chronic stress increased the density of neuron fiber in the TME of both BALB/c and NSG TNBC mice. Notably, the treatment of Propranolol (Prop, a β1- and β2-adrenergic receptor inhibitor) significantly decreased the tumor burden and metastasis of both BALB/c mice and NSG TNBC mice model. Accompany by recovering the function and number of TCD8+ cells in the TME of BALB/c mice. Conclusion: Our study indicates that chronic stress promotes TNBC progression and metastasis by regulating the tumor-immune network in the TME. Citation Format: Chao Zhang, Lizhong Wang, Runhua Liu. Chronic stress promotes tumor progression and metastasis through the tumor microenvironment in triple-negative breast cancer [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 2507.

Abstract 3635: TBK1 inhibition potentiates the efficacy of AXL-targeted therapy by modulating tumor microenvironment in aggressive breast cancers

Abstract Introduction: Triple-negative and inflammatory breast cancer (TNBC and IBC) are the most aggressive breast cancer subtypes. Novel actionable targets and complementary therapies are critically needed. AXL, a receptor tyrosine kinase, drives pleiotropic phenotypes of TNBC and IBC aggressiveness. Targeting AXL reduces IBC tumor growth in vivo and inhibits polarization of M2 macrophages. To identify novel tumor microenvironment (TME) targets to enhance the efficacy of AXL-targeted therapy, we conducted a synthetic lethal kinome siRNA screening. We identified TBK1 as a candidate to synergize with AXL growth inhibition. TBK1 is a serine/threonine protein kinase that regulates innate, adaptive responses and antitumor immune responses. We hypothesized that inhibition of TBK1 enhances the antitumor effect of AXL-targeted therapy in aggressive breast cancers. Methods: We used knockdown, knockout (KO) and inhibitors to suppress AXL and TBK1 pathways and tested the synergistic effect of targeting AXL and TBK1 on the growth of human SUM149 and BCX010 IBC cells, and HS578T TNBC cells in vitro. To determine the synergistic effect of targeting both pathways in vivo, we inoculated control or TBK1 KO murine TNBC 4T1.2 cells into BALB/c mice. We assessed the activity of AXL inhibitor TP-0903 in reducing tumor growth. Using multicolor flow cytometry, we studied the effects of targeting AXL and TBK1 on the TME. We tested the activity of TP-0903 combined with a TBK1 inhibitor in another TNBC mouse model, E0771. We used RNA-sequencing and real-time PCR/Western blot to determine the molecular mechanisms of how TBK1 inhibition synergizes AXL-targeted therapy in these cancers. Results: Compared with AXL or TBK1 suppression alone, genomic or pharmacologic suppression of AXL and TBK1 significantly reduced the growth of SUM149, BCX010, and HST578T cells in vitro. In both 4T1.2 and E0771 syngeneic mouse models, TP-0903 was more active in reducing tumor growth in TBK1-inhibited tumors than in control tumors. Tumors with TBK1 inhibition and treated with TP-0903 had a significantly higher population of cytotoxic T cells than control tumors. Depletion of CD8+ T cells blocked the synergistic effect of targeting AXL and TBK1 pathways on reducing tumor growth, suggesting that cytotoxic T cells contributed to the anti-tumor synergy of targeting AXL and TBK1. Mechanistically, TBK1 induced M2 macrophage migration via IRF3-regulated CCL5 secretion in SUM149 and HS578T cells, and AXL KO attenuated the polarization and migration of M2 macrophages by inhibiting the CCR5/CCL5 axis. Conclusions: Targeting TBK1 enhances the efficacy of AXL-targeted therapy in aggressive breast cancer by suppressing the paracrine effect of CCR5/CCL5 axis. This combination represents a novel and effective therapy modulating the TME of aggressive breast cancer, which warrants further investigation in the clinical setting. Citation Format: Lan Phi, Takashi Semba, Ngu V. Trinh, Fang Zou, Jason M. Foulks, Steven L. Warner, Savitri Krishnamurthy, James P. Long, James M. Reuben, Debu Tripathy, Naoto T. Ueno, Xiaoping Wang. TBK1 inhibition potentiates the efficacy of AXL-targeted therapy by modulating tumor microenvironment in aggressive breast cancers. [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 3635.

Abstract 73: Blockade of p38 MAPK reduces the tumor-induced immune suppressive microenvironment in metastatic breast cancer

Abstract The ability of CD8+ T cells to mount an anti-tumor immune response is compromised by immune suppression in the tumor microenvironment (TME). Tumor Associated Macrophages (TAMs) and Myeloid Derived Suppressor Cells (MDSCs) are a major part of this immune suppressive network. Targeting these populations remains challenging. Previously, we have reported that pharmacological and genetic blockade of p38 MAPK impeded the expansion and mobilization of monocytic and granulocytic MDSCs in mouse mammary carcinoma models. We also found that blockade of p38 or depletion of MDSCs reduced tumor growth and metastasis while enhancing the levels of CD8+ T cells in the primary tumors. In the present study, we asked whether CD8+ T cells contribute to the anti-metastatic activity of p38 inhibitor (p38i) and how p38 blockade affects the functional status of T cells and MDSCs. By using the mouse mammary carcinoma 4T1 model, we found that depletion of CD8+ T cells negated the effects of p38i on tumor growth and metastasis, indicating that CD8+ T cells contribute to the anti-tumor and anti-metastatic effects of p38 blockade. Next, we examined whether p38i exhibits a direct effect on T cells. The results of the T cell proliferation in vitro assays revealed that p38 blockade did not have a direct impact on T cell proliferation in response to αCD3/αCD28 stimulation. To determine the effect of p38 blockade on T cells in vivo, we performed single cell RNA-seq on the 4T1 tumor models treated with p38i and the 4T1 model with p38α (Mapk14) knockout (p38ko). This study revealed that p38 blockade by p38i or by inactivation of p38 in tumor cells decreased the amount of exhausted T cells and increased Th1 cells in the TME, indicating a positive effect on T cell functions. Furthermore, we observed a significant decrease in inflammatory signaling in granulocytes and monocytes upon p38 blockade. Our previous study showed that p38i did not affect generation of MDSCs in vitro in response to G-CSF & GM-CSF. To determine whether p38i alters MDSCs in vivo, we assessed MDSC gene signature in monocytic and granulocytic MDSCs isolated from spleens of tumor-bearing mice subjected to p38 blockade. This work revealed that the MDSC gene signature was reduced in both p38i and p38ko groups compared to tumor bearing mice treated with vehicle-control. These results indicated a reduction in the MDSC generation in the in vivo model. Our study revealed that blockade of p38 reduces tumor induced immune suppression and may enhance anti-tumor immune response in metastatic breast cancer. Citation Format: Priyanka Rajan, Justin Zonneville, Robert Zollo, Mackenzie Honikel, Sofija Raudins, Sean Colligan, Brian Morreale, Mohammed Alruwaili, Mohammed Alqarni, Scott Olejniczak, Joseph Barbi, Scott Abrams, Andrei Bakin. Blockade of p38 MAPK reduces the tumor-induced immune suppressive microenvironment in metastatic breast cancer [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 73.

Abstract 2360: Digital spatial profiling of RNA and protein in the tumor microenvironment of invasive lobular breast cancer

Abstract Invasive lobular breast cancer (ILC) is an understudied breast cancer subtype with late recurrence, metastasis to serosal surfaces including the peritoneum, and dismal long-term outcome. The interaction between a tumor and its microenvironment leads to phenotypic changes in stromal cells and the ECM that promote proliferation and invasion of malignant cells. ILC is histologically distinct from invasive ductal carcinoma, characterized by discohesive E-cadherin-negative tumor cells that grow in single file. We expect the TME to be unique in ILC. We hypothesized that differing levels of nuclear receptor expression in tumor cells would impact stromal cell composition, presumably through paracrine signaling. There is a range of glucocorticoid receptor (GR) expression in ILC. We sought to determine how crosstalk between GR+ or GR- ILC cells and their respective TME differentially impact stromal cell gene and protein expression, as well as the immune cell milieu. To profile the tumor and stroma separately, we performed nanoString GeoMx digital spatial profiling (DSP) of RNA and protein expression in GR-positive (+) and GR-negative (-) primary ILC. We performed RNA DSP for 12 primary ILCs: 5 strongly positive for GR, 5 GR- and 2 tumors with mixed GR expression. We also completed DSP of proteins on adjacent tissue sections for a total of 87 proteins. To profile tumor and TME independently we segmented regions into PanCK+ tumor and PanCK- stroma. RNA expression analyses revealed striking differences between GR+ and GR- ILC in both tumor cells and TME. Pathway analyses showed enrichment of genes encoding eukaryotic translation initiation and control factors and ribosomal proteins in GR+ tumor cells, as well as downregulation of immune response and antigen presentation pathways. In the TME of the GR- tumors, we saw significantly higher expression of collagen biosynthesis and extracellular matrix genes. Using a spatial deconvolution tool, we observed gene expression indicating markedly higher abundance of macrophages and other myeloid subsets, endothelial cells and fibroblasts in GR+ ILC stroma. There was also evidence for more regulatory T-cells, suggestive of an immune suppressive microenvironment in GR+ ILC. Protein DSP revealed upregulation of proteins involved in DNA repair and cell survival, and reduction of some immune checkpoint proteins in the GR+ tumor cells. In the TME of GR+ ILC, we observed more Ki-67 and ER-alpha as well as proteins expressed on T-regs, as well as downregulation of cytotoxic proteins such as granzyme A. We conclude that in-depth examination of molecular profiles in both the tumor cells and the TME of ILCs will give insight into how tumor GR expression/activation influences the crosstalk between ILC cells and their microenvironment. As expected, we observed heterogeneity between patients, but DSP will allow in-depth characterization of individual tumors at the molecular level. Citation Format: Lynda B. Bennett, Sunati Sahoo, Cheryl M. Lewis, Indu Raman, Candace Frerich, Guanchun Chen, Min Xu, Suzanne D. Conzen. Digital spatial profiling of RNA and protein in the tumor microenvironment of invasive lobular breast cancer [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 2360.

Abstract 3646: New functional role of tamoxifen in breast cancer immunomodulation: role of RACK7 in activation of type I interferon signaling and CEACAM1/TIM-3-dependent immunosuppression

Abstract Despite advancements of immunotherapy against various cancers, breast cancer still retains poor response to immune checkpoint blockade (ICB) therapy. Therefore, the identification of promising target or new strategy to enhance ICB therapy in breast cancer is crucial. Here, we uncover that the DNA-damaging potential of tamoxifen (TAM) can shape the unfavorable but ready-to-fire tumor immune microenvironment in breast cancer. We discover that a long-term TAM administration unexpectedly induces JAK/STAT signaling and Type I interferon (IFN)-stimulated gene expression to promote T-cell infiltration. Furthermore, TAM also induces the expression of CEACAM1, which acts as an alternative T-cell inhibitory ligand via binding to TIM-3, a “checkpoint” receptor expressed on CD4+ and CD8+ T cells. The chromatin ‘‘reader’’ RACK7/ZMYND8, which functions as a transcriptional repressor of IFN-stimulated gene (ISG) and a critical factor in DNA repair, is found to be downregulated upon tamoxifen exposure and involved in the tamoxifen-mediated cellular modulation. We demonstrate that TAM in conjunction with RACK7-knockdown (KD) triggers robust upregulation of ISGs and CEACAM1 in both estrogen receptor-positive (ER+) and triple negative breast cancer (TNBC) cells. This immunomodulatory effect lead by loss of RACK7 is specific to TAM treatment, and is not observed when combined with other endocrine therapeutics. TAM combined with RACK7-KD promotes mitochondrial DNA damage, which leads to accumulation of cytosolic DNA and subsequent activation of the cGAS/STING pathway. The murine breast orthotopic models with TS/A, EO771 and 4T1 cells further demonstrate that TAM-mediated immunomodulatory in conjunction with RACK7-KD evokes cytokine/chemokine secretion and further induces T-cell infiltration into tumor microenvironment. However, the tumor killing effect is limited due to promotion of T-cell exhaustion from CEACAM1-TIM-3 interaction between tumor and T cells. Thus, our study indicates that targeting CEACAM1-TIM-3 interaction is crucial for TAM-mediated tumor immune response. This brings promising therapeutic approach with TAM by combining RACK7-KD with blockage to CEACAM1-TIM-3 interaction in breast cancer. The resistance of tamoxifen treatment may be overcome, and RACK7 may serve as both a therapeutic target and a biomarker to enable ICB therapy. Citation Format: Marvin A. Aberin, Yu-Ling (Pony) Lee, Xin-Guo Hsu, Chen-Yang Shen, Yao-Ming Chang, Kun-Yuan Lin, Chandan Guha, Shu-Ping Wang. New functional role of tamoxifen in breast cancer immunomodulation: role of RACK7 in activation of type I interferon signaling and CEACAM1/TIM-3-dependent immunosuppression. [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 3646.

Abstract 3629: Calcium signalling and the breast cancer microenvironment

Abstract Calcium is a key regulator of a variety of pathways important in breast cancer progression, including those involved in cell proliferation and invasiveness. Calcium signaling is implicated in processes relevant to the breast cancer tumor microenvironment and altered calcium influx is a feature of cancer associated fibroblasts. In these studies, simultaneous use of genetically encoded calcium indicators with distinct fluorescence spectral properties (GCaMP6m or JRCaMP1b), were used to explore the cross-talk between breast cancer cells and fibroblasts via calcium signaling and changes in different cellular compartments. HMF3S human fibroblast cells co-cultured with breast cancer cells expressing GCaMP6m or JRCaMP1b were assessed in 2D and 3D co-culture using automated epi-fluorescence or confocal microscopy. Mitochondrial calcium levels were assessed using targeted GCaMP6m (2mtGCaMP6m) in HMF3S cells expressing JRCaMP1b. Results showed distinct spatial and temporal changes in free calcium levels between both fibroblasts and breast cancer cells and the mitochondria and cytosol during activation. These methodological advances provide an opportunity to better understand calcium signaling in cancer associated fibroblasts. These techniques will also help identify the calcium channels and pumps that could be targeted to manipulate the breast cancer tumor microenvironment to inhibit pathways important in breast cancer progression. Citation Format: Alice H. Bong, Krystyna A. Gieniec, Francisco Sadras, Mélanie Robitaille, Sarah J. Roberts-Thomson, Felicity M. Davis, Gregory R. Monteith. Calcium signalling and the breast cancer microenvironment. [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 3629.

KIF2C is a prognostic biomarker associated with immune cell infiltration in breast cancer

BackgroundThe kinesin-13 family member 2C (KIF2C) is a versatile protein participating in many biological processes. KIF2C is frequently up-regulated in multiple types of cancer and is associated with cancer development. However, the role of KIF2C in immune cell infiltration of tumor microenvironment and immunotherapy in breast cancer remains unclear.MethodsThe expression of KIF2C was analyzed using Tumor Immune Estimation Resource (TIMER) database and further verified by immunohistochemical staining in human breast cancer tissues. The correlation between KIF2C expression and clinical parameters, the impact of KIF2C on clinical prognosis and independent prognostic factors were analyzed by using TCGA database, the Kaplan-Meier plotter, and Univariate and multivariate Cox analyses, respectively. The nomograms were constructed according to independent prognostic factors and validated with C-index, calibration curves, ROC curves, and decision curve analysis. A gene set enrichment analysis (GSEA) was performed to explore the underlying molecular mechanisms of KIF2C. The degree of immune infiltration was assessed by the Estimation of Stromal and Immune cells in Malignant Tumor tissues using the Expression (ESTIMATE) algorithm and the single sample GSEA (ssGSEA). The Tumor mutational burden and Tumor Immune Dysfunction and Rejection (TIDE) were used to analyze immunotherapeutic efficiency. Finally, the KIF2C-related competing endogenous RNA (ceRNA) network was constructed to predict the putative regulatory mechanisms of KIF2C.ResultsKIF2C was remarkably up-regulated in 18 different types of cancers, including breast cancer. Kaplan-Meier survival analysis showed that high KIF2C expression was associated with poor overall survival (OS). KIF2C expression was associated with clinical parameters such as age, TMN stage, T status, and molecular subtypes. We identified age, stage, estrogen receptor (ER) and KIF2C expression as OS-related independent prognosis factors for breast cancer. An OS-related nomogram was developed based on these independent prognosis factors and displayed good predicting ability for OS of breast cancer patients. Finally, our results revealed that KIF2C was significantly related to immune cell infiltration, tumor mutational burden, and immunotherapy in patients with breast cancer.ConclusionKIF2C was overexpressed in breast cancer and was positively correlated with immune cell infiltration and immunotherapy response. Therefore, KIF2C can serve as a potential biomarker for prognosis and immunotherapy in breast cancer.

Abstract 1365: The spatially resolved host-microbiome sequencing platform reveals the interaction between bacteria and cancer stem cell microinches

Abstract The tumor microbiome is a unique element that appears differently for each type of cancer in a person. Currently, intratumoral host-microbiome sequencing was only done by bulk tissue analysis which means little is understood when it comes to the spatial distribution of microbiota in the heterogeneous tumor microenvironment and the localized effect of the microbiota. Breast cancer is known to be one of the cancers rich in the microbiome. We focused on cancer stem cell-like microniches related to the tumor microenvironment of triple-negative breast cancer(TNBC). We adapted SLACS(Spatially resolved Laser activated Cell Sorter) which isolates the region of interest from immunofluorescence-stained samples using a laser, providing the spatial distribution of localized microbiota in rare cancer stem cell samples. By applying SLACS to point out the breast cancer stem cell micro-niches, we discovered that more microbes flourished in ALDH1 high/CD44 high groups rather than normal cells. We also identified the composition of the microbiota was different in cancer stem cells depending on the degree of expression of the marker. Comparing RNA-seq data of host and microbiome sequencing with functional studies, we reveal the distribution of microbiota in breast tumors and interactions between cancer stem cells and microbiota. Collectively, we found the spatial pattern of the microbiota localized in cancer stem cell microniches and how cells and bacteria interact with each other more precisely. Citation Format: Gyeongjun Kim. The spatially resolved host-microbiome sequencing platform reveals the interaction between bacteria and cancer stem cell microinches [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 1365.

Comprehensive analysis of glycolysis mediated pattern clusters and immune infiltration characterization of tumor microenvironment in triple-negative breast cancer

BackgroundThe involvement of glycolysis in carcinogenesis and the tumor microenvironment is being increasingly supported by the available data. The aim of this work was to develop a triple-negative breast cancer predictive model based on glycolysis. MethodsGlycolysis mediated pattern clusters were created using the R “ConsensusClusterPlus” package. The variations in the tumor microenvironment between the pattern clusters were examined using the R “GSVA”, “ESTIMATE”, and “CIBERSORT” package. The risk score and nomogram were established to assess clinical outcomes of patients. ResultsSubstantial differences were observed in the immunological landscape between the glycolysis-mediated pattern clusters. When it came to predicting survival and immunotherapy response, the developed risk score showed promising predictive power. Nomogram was designed to be used in therapeutic settings due to its remarkable predictive accuracy. ConclusionsThe immune microenvironment varied among cases of triple-negative breast cancer. The nomogram and the risk score based on glycolysis could both be used to help create more effective treatments.

RISING STARS: Hormonal regulation of the breast cancer microenvironment.

The tumor microenvironment is a dynamic ecosystem of stromal and immune cells that, under the influence of cancer cells, govern biochemical signaling, mechanical signaling via production and remodeling of the extracellular matrix (ECM), formation of vascular networks, and ultimately promotion of tumor growth. In breast cancer, hormone receptor-mediated signaling is a key coordinator of cancer cell proliferation and invasiveness not only through cell-autonomous means but also via cancer cell-stroma cross-talk. In the absence of hormone receptors, a different microenvironment landscape emerges, which comes with its own challenges for therapy. This review summarizes the current knowledge regarding the associations of hormone receptor profiles with composition of the microenvironment, how hormones directly influence stromal cells, immune cells and cells associated with the vasculature, and the paracrine mechanisms that lead to the formation of a tumor-promoting ECM.

Panax notoginseng saponins normalises tumour blood vessels by inhibiting EphA2 gene expression to modulate the tumour microenvironment of breast cancer

BackgroundPanax notoginseng saponins (PNS), the main active component of Panax notoginseng, can promote vascular microcirculation. PNS exhibits antitumor effects in various cancers. However, the molecular basis of the relationship between PNS and tumor blood vessels remains unclear. PurposeTo study the relationship between PNS inhibiting the growth and metastasis of breast cancer and promoting the normalization of blood vessels. MethodsWe performed laser speckle imaging of tumor microvessels and observed the effects of PNS on tumor growth and metastasis of MMTV-PyMT (FVB) spontaneous breast cancer in a transgenic mouse model. Immunohistochemical staining of Ki67 and CD31 was performed for tumors, scanning electron microscopy was used to observe tumor vascular morphology, and flow cytometry was used to detect tumor tissue immune microenvironment (TME). RNA-seq analysis was performed using the main vessels of the tumor tissues of the mice. HUVECs were cultured in tumor supernatant in vitro to simulate tumor microenvironment and verify the sequencing differential key genes. ResultsAfter treatment with PNS, we observed that tumor growth was suppressed, the blood perfusion of the systemic tumor microvessels in the mice increased, and the number of lung metastases decreased. Moreover, the vascular density of the primary tumor increased, and the vascular epidermis was smoother and flatter. Moreover, the number of tumor-associated macrophages in the tumor microenvironment was reduced, and the expression levels of IL-6, IL-10, and TNF-α were reduced in the tumor tissues. PNS downregulated the expression of multiple genes associated with tumor angiogenesis, migration, and adhesion. In vitro tubule formation experiments revealed that PNS promoted the formation and connection of tumor blood vessels and normalized the vessel morphology primarily by inhibiting EphA2 expression. In addition, PNS inhibited the expression of tumor vascular marker proteins and vascular migration adhesion-related proteins in vivo. ConclusionIn this study, we found that PNS promoted the generation and connection of tumor vascular endothelial cells, revealing the key role of EphA2 in endothelial cell adhesion and tumor blood vessel morphology. PNS can inhibit the proliferation and metastasis of breast cancer by inhibiting EphA2, improving the immune microenvironment of breast cancer and promoting the normalization of tumor blood vessels.

NAD+ biosynthesis metabolism predicts prognosis and indicates immune microenvironment for breast cancer.

The growing evidence implies that tumor cells need to increase NAD+ levels by upregulating NAD+ biosynthesis to satisfy their growth demand. NAD+ biosynthesis metabolism is implicated in tumor progression. Breast cancer (BC) is the most common malignant malignancy in the world. Nevertheless, the prognostic significance of NAD+ biosynthesis and its relationship with the tumor immune microenvironment in breast cancer still need further investigation. In this study, we obtained the mRNA expression data and clinical information of BC samples from public databases and calculated the level of NAD+ biosynthesis activity by single-sample gene set enrichment analysis (ssGSEA). We then explored the relationship between the NAD+ biosynthesis score, infiltrating immune cells, prognosis significance, immunogenicity and immune checkpoint molecules. The results demonstrated that patients with high NAD+ biosynthetic score displayed poor prognosis, high immune infiltration, high immunogenicity, elevated PD-L1 expression, and might more benefit from immunotherapy. Taken together, our studies not only deepened the understanding of NAD+ biosynthesis metabolism of breast cancer but also provided new insights into personalized treatment strategies and immunological therapies to improve the outcomes of breast cancer patients.

Role of Gut Microbiota in Breast Cancer and Drug Resistance.

Breast cancer is the most common malignancy in women worldwide. The cause of cancer is multifactorial. An early diagnosis and the appropriate treatment of cancer can improve the chances of survival. Recent studies have shown that breast cancer is influenced by the microbiota. Different microbial signatures have been identified in the breast microbiota, which have different patterns depending on the stage and biological subgroups. The human digestive system contains approximately 100 trillion bacteria. The gut microbiota is an emerging field of research that is associated with specific biological processes in many diseases, including cardiovascular disease, obesity, diabetes, brain disease, rheumatoid arthritis, and cancer. In this review article, we discuss the impact of the microbiota on breast cancer, with a primary focus on the gut microbiota's regulation of the breast cancer microenvironment. Ultimately, updates on how immunotherapy can affect the breast cancer-based microbiome and further clinical trials on the breast and microbiome axis may be an important piece of the puzzle in better predicting breast cancer risk and prognosis.

A positive feedback loop driven by fibronectin and IL-1β sustains the inflammatory microenvironment in breast cancer

Inflammatory alterations of the extracellular matrix shape the tumor microenvironment and promote all stages of carcinogenesis. This study aims to determine the impact of cellular fibronectin on inflammatory facets of tumor-associated macrophages (TAMs) in breast cancer. Cellular fibronectin (FN) harboring the alternatively spliced extra domain A (FN-EDA) was determined to be a matrix component produced by the triple-negative breast cancer (TNBC) cells. High levels of FN-EDA correlated with poor survival in breast cancer patients. The proinflammatory cytokine IL-1β enhanced the expression of cellular fibronectin including FN-EDA. TAMs were frequently observed in the tumor areas rich in FN-EDA. Conditioned media from TNBC cells induced the differentiation of CD206+CD163+ macrophages and stimulated the STAT3 pathway, ex vivo. In the macrophages, the STAT3 pathway enhanced FN-EDA-induced IL-1β secretion and NF-κB signaling. In conclusion, our data indicate a self-reinforcing mechanism sustained by FN-EDA and IL-1β through NF-κB and STAT3 signaling in TAMs which fosters an inflammatory environment in TNBC.

P054 Molecular characterization and the role of lymphocyte microenvironment in breast cancer

P054 Molecular characterization and the role of lymphocyte microenvironment in breast cancer

SA 3.2 Tumor microenvironment in early breast cancer

SA 3.2 Tumor microenvironment in early breast cancer

An Angiogenesis-Related lncRNA Signature Is Associated with Prognosis and Tumor Immune Microenvironment in Breast Cancer.

Angiogenesis is crucial in the development and progression of tumors. This study examined the relationship between angiogenesis-related lncRNAs (AR-lncRNAs) and breast cancer (BC) immunity and prognosis. We used univariate Cox regression analysis to obtain AR-lncRNAs closely related to BC prognosis. Cluster analysis of BC patients was performed using non-negative matrix factorization (NMF) analysis according to the expression of AR-lncRNAs that were prognostically relevant. An AR-lncRNA risk model (AR-lncM) was created using LASSO regression analysis to predict the prognosis and survival of BC patients. Subsequently, the effect of LINC01614 on cell migration and invasion was verified by Transwell and Western blot assays, and the CCK-8 assay detected its impact on cell sensitivity to tamoxifen. Finally, we obtained 17 AR-lncRNAs from the TCGA database that were closely associated with the prognosis of BC patients. Based on the expression of these AR-lncRNAs, BC patients were divided into five clusters using NMF analysis. Cluster 1 was found to have a better prognosis, higher expression of immune checkpoints, and higher levels of immune cell infiltration. Furthermore, an AR-LncM model was created using ten prognostic-related AR-lncRNAs. The model's risk predictive performance was validated using survival analysis, timeROC curves, and univariate and multivariate Cox analysis. The most interesting gene in the model, LINC01614, was found to regulate epithelial-mesenchymal transition (EMT) and tamoxifen sensitivity in BC cells, implying that LINC01614 could be a potential therapeutic target for BC patients.