Murine Breast Tumor Research Articles

Abstract 1476: Vessel normalization following LDH-A knockdown in murine breast tumors

Abstract Abnormal vessel formation plays a major role in tumor progression. Previously, we showed that lactate dehydrogenase A (LDH-A) knockdown (KD) reduces orthotopic 4T1 breast tumor lactate, and has a robust anti-tumor effect through changes in the tumor microenvironment (TME) [1, 2]. Tumors with LDH-A downregulation have lower VEGF-A secretion and reduced formation of new blood vessels [2]. In addition, LDH-A KD cells/tumors displayed reduced HIF-1 signaling, as assessed by BLI using HRE-exGLuc reporter. Changes in the bioluminescence signal corresponded with changes in HIF-1α staining. LDH-A KD tumors also showed less HIF-1α staining compared to control tumors, consistent with less pimonidazole (hypoxia) staining [2]. Murine 4T1 breast cancer cells and tumors with LDH-A knockdown were developed and described [2]. To evaluate the degree of tumor neovascularization and its normalization, we stained tumors for CD31 (endothelial marker) and NG2 (pericytes marker). For pericyte coverage quantification, vessels were counted, and percentage of vessels covered by pericytes were calculated. Blood vessel permeability was assessed by intravenous injection of Evans Blue dye and tumor dye extravasation was quantified. To assess in vivo vascular network over time, a raster-scan optoacoustic mesoscopy (RSOM) was performed. Vessel structure in 4T1 murine tumors changes significantly following LDH-A KD. Normalized LDH-A KD tumor neovascularization was shown using the double immunofluorescence staining for CD31 and NG2, including a more normal vascular morphology, with shorter, more linear (less branched) vessel structure, with a higher percentage of vessels covered by pericytes compared to control tumors. Evans Blue dye extravasation was significantly less (lower vascular permeability) in LDH-A KD tumors compared to control tumors. Applying RSOM to follow the development of the vascular network during tumor growth, we showed better vascularization of LDH-A KD tumors. Furthermore, the number of circulating tumor cells (CTCs) was also significantly lower in blood collected from mice bearing LDH-A KD tumors. We show that LDH-depletion leads to tumor vessel normalization that includes several unique features: structural changes, greater coverage of blood vessel by pericytes and reduced vessel leakiness, leading to changes in the tumor microenvironment, inhibition of metastases formation and prolongation of survival. 1. Rizwan, A., et al., Relationships between LDH-A, lactate, and metastases in 4T1 breast tumors. Clin Cancer Res, 2013. 19(18): p. 5158-69. 2. Serganova, I., et al., LDH-A regulates the tumor microenvironment via HIF-signaling and modulates the immune response. PLOS ONE, 2018. 13(9): p. e0203965. Citation Format: Inna Serganova, Kiranmayi Vemuri, Ivan Cohen, Matthew Lubin, Sheryl Roberts, Masatomo Maeda, Mayuresh Mane, Henry Mann, Thomas Reiner, Eric Chan, Dmitry Yarilin, Katia Manova-Todorova, Roberta Zappasodi, Taha Merghoub, Jason Koutcher, Ronald Blasberg. Vessel normalization following LDH-A knockdown in murine breast tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1476.

Abstract 2206: Degradation of hyaluronan in the tumor microenvironment enhances oncolytic reovirus and anti-PD-L1 therapy in a murine syngeneic breast tumor model

Abstract The extracellular matrix (ECM) structure heavily influences tumor growth, invasion and various steps of the cancer-immunity cycle. Hyaluronan (HA), a major component of ECM, is a glycosaminoglycan that accumulates in the tumor microenvironment (TME) of many solid tumor types, and HA accumulation can restrict T cell infiltration and promote immunosuppression. We have demonstrated in preclinical tumor models that enzymatic degradation of TME HA by intravenous PEGylated recombinant human hyaluronidase PH20 (Pegvorhyaluronidase alfa, PEGPH20) facilitates delivery of chemotherapeutics to tumors and enhances efficacy of current immunotherapy strategies including checkpoint inhibitors. In this study, we hypothesized that degradation of HA with PEGPH20 treatment may remodel the TME to improve the efficacy of oncolytic reovirus therapy (pelareorep) and checkpoint blockade therapy. To test this hypothesis, EMT6 murine breast tumor cells, which spontaneously accumulate HA and are sensitive to oncolytic activity of pelareorep were implanted orthotopically into the mammary fat pad of BALB/c mice. In this model, PEGPH20 treatment resulted in a reduction of HA measured by immunohistochemistry and induced tumor growth inhibition (30% TGI). Pelareorep plus anti-PD-L1 treatment also resulted in anti-tumor activity (44% TGI), and flow cytometric analysis of tumor infiltrating immune cells in pelareorep + anti-PD-L1-treated tumors demonstrated a significant increase in the numbers of cytotoxic CD8+ tumor infiltrating lymphocytes (TILs, p=0.0004), a decrease in regulatory T cells (T reg, p<0.0001) and an increase in CD8:Treg ratio (p=0.0018), indicating a robust antitumor immune response. Combination of PEGPH20 with pelareorep + anti-PD-L1 resulted in enhanced anti-tumor activity (71% TGI) compared to either PEGPH20 (30%TGI) or pelareorep + anti-PD-L1 (44% TGI). PEGPH20 in combination with pelareorep + anti-PDL1 also extended survival (median survival time, MST- 30 days, p<0.0001) in comparison with PEGPH20 alone (MST-21 days) or pelareorep + anti-PD-L1 (MST-22 days). Our data demonstrate the potential for matrix modeling through degradation of HA as a strategy to improve oncolytic reovirus + anti-PD-L1 immunotherapy. These findings are consistent with prior preclinical studies and support clinical evaluation of combining PEGPH20 with immune checkpoint inhibitors in HA-accumulating tumors. Citation Format: Jisook Lee, Grey A. Wilkinson, Trevor Kimbler, Barbara Blouw, Curtis B. Thompson. Degradation of hyaluronan in the tumor microenvironment enhances oncolytic reovirus and anti-PD-L1 therapy in a murine syngeneic breast tumor model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2206.

Abstract 5606: Characterization of temporal proteomic dynamics of murine breast and melanoma tumor microenvironments without and with pulsed focused ultrasound

Abstract Focused ultrasound (FUS) ablation has been successfully used for thermal ablation of solid malignancies. However, non-ablative pulsed-FUS (pFUS) has received less attention but could be less destructive anti-tumor therapy by stimulating a pro-inflammatory immune response within the tumor microenvironment (TME). Clinically, such an approach could be useful to treat peripheral disease (e.g. micro-metastases) in otherwise normal-appearing tissues. Here, we explored the alterations of cytokines, chemokines and trophic factors (CCTF) and cell adhesion molecules (CAM) in the TME of mouse flank tumor models following pFUS treatment at different time points of tumor progression. Mice were bilaterally inoculated with subcutaneous B16 melanoma or 4T1 breast tumors into the hind limbs and allowed to reach diameters of ~5 mm (n=6/group/timepoint) before performing experiments (Day 1). Separate groups of mice were subjected to pFUS (1 MHz; Peak negative pressure = 6 MPa; Duty cycle = 10%, 100 pulses per site) at Days 1, 5, or 10 and euthanized 24 hr post-pFUS for proteomic analyses. Separate groups of time-matched tumor-bearing mice that did not receive pFUS were also euthanized at the same time points both to assess relative changes due to pFUS at each time point and provide information regarding the natural history of TME progression in each tumor type. Proteomic analyses in untreated control tumors revealed an overall shift toward immunosuppressive (pro-tumor) TME over the 10 days. When comparing proteomic effects of pFUS treatment in compared to untreated time-matched controls, tumors treated on Day 1 exhibited greater upregulation of pro-inflammatory (anti-tumor) CCTF and CAM and pFUS had less pronounced effects on the TME when administered in groups of mice treated at Days 5 or 10. To further examine the acute effects of pFUS at on CCTF and CAM over 72hr. pFUS treatment in elicited unique CCTF and CAM profiles for the two tumor types, but both could be considered shifted towards anti-tumoral TME, especially within the first 24 hrs. These data suggest that pFUS can potentially modulate TME in a therapeutically beneficial way. However, they reveal two important caveats that require substantial investigation: 1) Different tumor types have unique molecular responses and imply pFUS may be more suitable for certain tumor types; 2) Natural histories encompass dynamic TME changes as part of disease progression and appropriate timing of pFUS in each tumor type will be necessary to maximizing the potential therapeutic benefits of pFUS. Citation Format: Gadi Cohen, Parwathay Chandran, Lauren E. Tomlinson, Maggie E. Sundby, Rebecca M. Lorsung, Scott R. Burks, Joseph A. Frank. Characterization of temporal proteomic dynamics of murine breast and melanoma tumor microenvironments without and with pulsed focused ultrasound [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5606.

Abstract 5694: Complete regression of murine breast tumors by hetIL-15FC monotherapy correlates with infiltration by T, NK, cDC1 cells and a novel population of dendritic cells

Abstract Introduction: IL-15 cytokine stimulates the proliferation and cytotoxic function of CD8+ T and NK cells. Several preclinical models have supported the anti-tumor activity of IL-15. We have produced the native heterodimeric IL-15 (hetIL-15) of human, macaque or mouse and also fusions to the Fc antibody fragments (hetIL-15FC). Experimental procedures: We studied the therapeutic efficacy of hetIL-15 or hetIL-15FC immunotherapy in the murine EO771 orthotopic breast cancer model in syngeneic C57BL/6 mice. The effects of hetIL-15FC on immune cells were compared in tumors, lymph nodes and spleen by flow cytometry, immunohistochemistry (IHC) and gene expression profiling (Nanostring). We also assessed the metabolic profile of tumor infiltrating T cells. Results: hetIL-15 therapy delayed the primary tumor growth and increased overall survival. hetIL-15 or hetIL-15FC peritumoral administration resulted in tumor regression in 40% of the treated animals and resistance to tumor re-challenge. Therefore, monotherapy induced long term immunological memory able to block tumor growth. Both CD8+ T and NK cells were increased in hetIL-15FC treated tumors; phenotypic analysis showed they were activated and in an active proliferation state. Transcriptomic analysis confirmed the activated state of the T and NK cells, whereas metabolic flux analysis of the infiltrated CD8+T cells from treated mice confirmed a rise in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity (as a measure of mitochondrial reserve), which supports an activated/non exhausted phenotype of these hetIL-15 treated effector cells. In addition, peritumoral hetIL-15FC administration resulted in an increase of infiltrating, conventional type 1 dendritic cells (cDC1s), suggesting mechanisms of DC-lymphocyte interactions in the tumor. We also observed a new population of intra-tumoral DCs induced by hetIL-15, CD24+ F4/80high cDC2s (F4/80high DCs). The new DC population was negatively correlated with the tumor size of EO771 tumor bearing mice. Phenotypic profiling of F4/80high DCs identified expression of several cDC1 specific markers, including CD103 and IRF8 and t-SNE analysis clustered F4/80high DCs close to cDC1s and macrophages. Conclusion: Our results show complete eradication of EO771 tumors by hetIL-15FC treatment, correlating with different tumor infiltrating immune cell types. We propose that the anti-cancer activity of hetIL-15 in primary tumors is orchestrated by the interplay of CD8+T cells, cDC1s and a novel subset of antigen presenting cells with a distinct phenotypic profile. hetIL-15 supported a favorable metabolic profile of intra-tumoral effector lymphocytes, important for their function. Effective hetIL-15 treatment leads to development of long-term anti-tumor memory. Citation Format: Sevasti Karaliota, Dimitris Stellas, Vasiliki Stravokefalou, Bethany Nagy, Cristina Bergamaschi, Barbara K. Felber, George N. Pavlakis. Complete regression of murine breast tumors by hetIL-15FC monotherapy correlates with infiltration by T, NK, cDC1 cells and a novel population of dendritic cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5694.

Abstract 2770: Imaging cancer immunology: Monitoring CD47 mAb treatment in vivo by magnetic particle imaging

Abstract Introduction: The rapid growth of research into immuno-oncology research has fueled a need to be able to evaluate the efficacy of immunotherapies in a timely and comprehensive fashion. However, established non-invasive imaging approaches have been insufficient to meet all requirements. Magnetic Particle Imaging (MPI) is a novel tomographic molecular imaging technique that can be used to track iron-oxide in 3D in vivo, with sensitivity and specificity similar to nuclear medicine but without the complex workflow, safety, and half-life limitations. Methods: A murine breast tumor model was established by injecting 3 × 105 4T1 cells into the 4th mammary fat pad of 8-10 week female Blab/c mice. A CD47 monoclonal antibody (mAb) treatment was initiated by intraperitoneal injection of CD47 mAb (Bio-X Cell) at day 6 after tumor implantation. The treated and the control groups were then injected with an iron-oxide MPI tracer (6mg/kg) and then 3D MPI images were acquired 8, 11, 14 and 17 days after implantation. µCT and MRI were also acquired with a T2-weighted multi-slice, multi-echo (MSME) sequences at above time points. Both MPI and MRI images were co-registered and quantified. Tumors, liver, spleen and draining lymph nodes were then harvested, imaged, fixed, and stained with Perls Prussian blue. Results: Superparamagnetic iron oxide nanoparticles accumulation has been shown qualitatively to increase following CD47 mAb treatment [1]. To determine iron accumulation within the tumor, MPI was co-registered to both MRI µCT for anatomical context. All mice showed an accumulation of nanoparticles in the tumor and liver following injection in both MRI and MPI. Quantitation of iron at the tumor was performed and compared between the CD47-treated and untreated groups at every imaging time point with MPI. CD47 mAb treated breast tumors demonstrated enhanced positive contrast on 3D MPI images while a shortened T2 relaxation on MRI images. Iron content within the tumor was confirmed by ex vivo scanning followed by staining. Conclusions: By combining the sensitivity, specificity and quantitation potentials of MPI, information can be obtained on a preclinical model that monitors the efficacy of CD47 mAb cancer immunotherapy. With the high spatial resolution provided by MRI, coregistered MRI-MPI can be utilized to evaluate the response of tumor-associated macrophages to CD47 mAb and other cancer immunotherapies.

MHealth: A smartphone-controlled, wearable platform for tumour treatment

With the booming development of smartphones in our daily lives, the integration of advanced medical technologies and mobile health (mHealth) to manage disease has become an irresistible trend. To extend quality tumour treatment into our daily lives, an mHealth platform was developed by using ferroelectric BiFeO3 nanomaterials (BFO) modified with TAT peptide (TAT-BFO) and a smartphone-controlled wearable device. With the control of a wearable device, TAT-BFO could be enriched in tumour site via magnetic targeting, and then triggered to generate reactive oxygen species (ROS) for efficient tumour therapy through temperature fluctuation. In both murine orthotopic breast tumour and resectable breast tumour models, this mHealth platform was demonstrated to inhibit tumour growth and prevent tumour recurrence significantly. Moreover, the transcriptomics and multiphysics simulation analysis further elucidated the working mechanism of the mHealth platform. This mHealth platform exhibits great potential for guiding daily-treatment and simplifying clinical treatment of tumours.

Immunomodulation via macrophages to fight solid tumor malignancies

The paper 'A C-Terminal Fragment of Adhesion Protein Fibulin-7 Inhibits Growth of Murine Breast Tumor by Regulating Macrophage Reprogramming' by Chakraborty et al. highlights that Fbln7-C could be explored as a potential immunomodulatory agent against various solid cancers and have shown its abilities to regulate tumor microenvironment reprogramming of TAMs in a breast cancer model. Fbln7, which is a secreted glycoprotein, has been shown to be anti-angiogenic and has an immunomodulatory role regulating various functional properties of monocytes, macrophages, and neutrophils, thereby influencing inflammation. In this study, the authors have shown that in a murine breast tumor model, intravenous administration of Fbln7-C significantly reduces the size of tumors via macrophage reprogramming. Comment on: https://doi.org/10.1111/febs.15333.

In Vitro and In Vivo Efficacy of AZD3965 and Alpha-Cyano-4-Hydroxycinnamic Acid in the Murine 4T1 Breast Tumor Model

Monocarboxylate transporter 1 (MCT1) represents a potential therapeutic target in cancer. The objective of this study was to determine the efficacy of AZD3965 (a specific inhibitor of MCT1) and α-cyano-4-hydroxycinnamic acid (CHC, a nonspecific inhibitor of MCTs) in the murine 4T1 tumor model of triple-negative breast cancer (TNBC). Expression of MCT1 and MCT4 in 4T1 and mouse mammary epithelial cells were determined by Western blot. Inhibition of MCT1-mediated L-lactate uptake and cellular proliferation by AZD3965 and CHC was determined. Mice bearing 4T1 breast tumors were treated with AZD3965 100mg/kg i.p. twice-daily or CHC 200mg/kg i.p. once-daily. Tumor growth, metastasis, intra-tumor lactate concentration, immune function, tumor MCT expression, and concentration-effect relationships were determined. AZD3965 and CHC inhibited cell growth and L-lactate uptake in 4T1 cells. AZD3965 treatment resulted in trough plasma and tumor concentrations of 29.1 ± 13.9 and 1670 ± 946nM, respectively. AZD3965 decreased the tumor proliferation biomarker Ki67 expression, increased intra-tumor lactate concentration, and decreased tumor volume, although tumor weight was not different from untreated controls. CHC had no effect on tumor volume and weight, or intra-tumor lactate concentration. AZD3965 treatment reduced the blood leukocyte count and spleen weight and increased lung metastasis, while CHC did not. These findings indicate AZD3965 is a potent MCT1 inhibitor that accumulates to high concentrations in 4T1 xenograft tumors, where it increases tumor lactate concentrations and produces beneficial effects on markers of TNBC; however, overall effects on tumor growth were minimal and lung metastases increased.

A C-terminal fragment of adhesion protein fibulin-7 inhibits growth of murine breast tumor by regulating macrophage reprogramming.

Recent reports have shown that a C-terminal fragment of adhesion protein Fibulin7 (Fbln7-C) could demonstrate both antiangiogenic and anti-inflammatory activities. The current study investigated the potential of Fbln7-C as a modulator of tumor-associated macrophages (TAMs) and its potential as an anticancer therapeutic. Our invitro data show that Fbln7-C could inhibit the tumor cell line (MDA-MB-231) supernatant-induced reprogramming of human monocytes into immunosuppressive TAMs as indicated by higher expression of pERK1/2 and pSTAT1 molecules, and reduced expression of CD206 protein and arg1, ido, and vegf transcripts in monocytes cultured in the presence of Fbln7-C compared to controls. Interestingly, Fbln7-C-treated macrophages retained their altered phenotype even after the removal of Fbln7-C, and their secretome demonstrated anticancer activities. Finally, in a 4T1-induced murine breast tumor model, intravenous administration of Fbln7-C, following the appearance of measurable tumors, significantly reduced the growth and weight of the tumors. Detailed phenotypic analysis of the infiltrated monocyte/macrophage populations (F480+ Ly6G- CD11b+ ) at day 23 postinduction showed a higher percentage of inflammatory monocytes (F480+ Ly6Chi CD11b+ ) and a delayed differentiation into anti-inflammatory TAMs as evident by their reduced levels of CD206 expression. In conclusion, the above data suggest that Fbln7-C could regulate the tumor environment-induced macrophage reprogramming and has the potential for cancer therapeutics.

Regression of murine breast tumors after hetIL-15 monotherapy correlates with a novel population of intratumoral dendritic cells, in addition to increased infiltration of T, NK and cDC1 cells

Abstract Background We studied mechanisms of therapeutic efficacy of the heterodimeric IL-15 (hetIL-15) of human, macaque or mouse in several mouse tumor models, including the murine EO771 orthotopic breast cancer model in syngeneic C57BL/6 mice. Methods The effects of hetIL-15 on immune cells were compared in tumors, lymph nodes and spleen by flow cytometry, immunohistochemistry (IHC), gene expression profiling (Nanostring) and metabolic profiling of tumor infiltrating T cells. Results hetIL-15 administration resulted in tumor regression in 40% of the treated animals and resistance to tumor rechallenge, inducing long term immunological memory. Activated NK and CD8+ T cells were increased in hetIL-15 treated tumors. hetIL-15 administration resulted in increased intratumoral infiltration of conventional Dendritic cells (cDC1) and also induced a new population of intratumoral DC, (F4/80high DCs), which were correlated negatively with the tumor size. Phenotypic profiling of F4/80high DCs identified expression of several cDC1 specific markers, including CD103, IRF8 and XCR1. tSNE analysis clustered F4/80high DCs close to cDC1s and macrophages. Conclusion Complete eradication of EO771 tumors by hetIL-15 treatment correlates with different tumor infiltrating immune cell types. We propose that the anti-cancer activity of hetIL-15 in primary tumors is orchestrated by the interplay of CD8+T cells, cDC1 and a novel subset of antigen presenting cells with a distinct phenotypic profile. Effective hetIL-15 treatment leads to development of long term anti-tumor memory. hetIL-15 supported a favorable metabolic profile of intratumoral effector lymphocytes, important for their function.

Green Tea Polyphenol EGCG Attenuates MDSCs-mediated Immunosuppression through Canonical and Non-Canonical Pathways in a 4T1 Murine Breast Cancer Model.

Several studies in the past decades have reported anti-tumor activity of the bioactive compounds extracted from tea leaves, with a focus on the compound epigallocatechin-3-gallate (EGCG). However, further investigations are required to unravel the underlying mechanisms behind the anti-tumor activity of EGCG. In this study, we demonstrate that EGCG significantly inhibits the growth of 4T1 breast cancer cells in vitro and in vivo. EGCG ameliorated immunosuppression by significantly decreasing the accumulation of myeloid-derived suppressor cells (MDSCs) and increasing the proportions of CD4+ and CD8+ T cells in spleen and tumor sites in 4T1 breast tumor-bearing mice. Surprisingly, a low dose of EGCG (0.5–5 μg/mL) effectively reduced the cell viability and increased the apoptosis rate of MDSCs in vitro. EGCG down-regulated the canonical pathways in MDSCs, mainly through the Arg-1/iNOS/Nox2/NF-κB/STAT3 signaling pathway. Moreover, transcriptomic analysis suggested that EGCG also affected the non-canonical pathways in MDSCs, such as ECM–receptor interaction and focal adhesion. qRT-PCR further validated that EGCG restored nine key genes in MDSCs, including Cxcl3, Vcan, Col4a1, Col8a1, Oasl2, Mmp12, Met, Itsnl and Acot1. Our results provide new insight into the mechanism of EGCG-associated key pathways/genes in MDSCs in the murine breast tumor model.

Abstract B45: NFAT5/TonEBP mediated anti-tumor efficiency of high salt activated CD4+T lymphocytes

Abstract Cell-based immunotherapy is rapidly emerging as a promising alternative to conventional chemotherapy-based treatment for cancer. Previous studies have shown that high salt treatment induces naive CD4+T lymphocytes to differentiate into a highly proinflammatory Th17 phenotype. In our current study, we analyzed the ability of salt treatment to induce cancer-specific Th17 lymphocytes. Naive CD4+T cells isolated by immunomagnetic bead isolation from splenocytes of 10-week-old BALB/c mice were treated in vitro with high salt (Δ0.05 M NaCl) along with CD3 monoclonal antibodies (mAbs), CD28 mAbs and IL-2 for 4 days along with heat killed 4T1 breast cancer cells. The isolated CD4+CCR6+CD45RA-CXCR3- cells were tested for IL-17 expression by flow cytometry and determined to be >95% pure. Orthotopic murine breast tumor models established by injection of 4T1 cells into the mammary fat pad of Nu/J mice were administered two doses of (1 × 106) salt induced Th17 cells on day 7 and day 12, post-tumor implantation. By day 28, the high salt induced Th17 cells cohort demonstrated significantly reduced tumor growth (97 ± 34 mm3 vs 512 ± 91 mm3, untreated control, p<0.05) and lung metastasis. However, no effect on tumor growth was noted when these 4T1 sensitized high salt treated Th17 cells were injected into Nu/J mice with prostrate (RM1) and lung tumors (KLN205), thus suggesting that the specificity of salt induced Th17 cells to breast tumors. Similarly, in vitro high salt mediated differentiation of Th17 cells when co-treated with heat killed RM1 and KLN205 cancer cells were able to induce respective murine tumor regression. Mechanistic studies demonstrated that the high salt induction to the Th17 phenotype was mediated by the tonicity-dependent transcription factor, TonEBP/NFAT5. The shRNA-based knockdown of TonEBP abrogated the induction of the Th17 phenotype and expression of proinflammatory cytokines IL-17, IL-1β and IL-6. Taken together, our data suggests that high salt mediated in vitro differentiation of Th17 cells could induce tumor specific anticancer CD4+T lymphocytes, which may give rise to a cell-based anticancer immunotherapy application. Citation Format: Suneetha Amara, Pooja Yenuga, Roy Zent, Venkataswarup Tiriveedhi. NFAT5/TonEBP mediated anti-tumor efficiency of high salt activated CD4+T lymphocytes [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 B45.

Abstract B97: The immunomodulatory roles of tumor-associated lymphatics in triple-negative breast cancer

Abstract During cancer development, growth and remodeling of lymphatic vessels—termed lymphangiogenesis—is associated with increased metastasis rate. However, our group has recently shown that lymphangiogenesis can also increase tumor immune infiltration and subsequently enhance the responsiveness to immunotherapies in melanoma. However, the role of lymphangiogenesis in antitumor immunity in breast cancer remains unexplored. Moreover, despite the success of immunotherapies in different cancer types, their translation into the treatment of breast cancer has remained challenging. Here, we investigated the role of tumor lymphangiogenesis in immune cell infiltration and responsiveness to immunotherapy in a murine triple-negative breast tumor model. Spontaneously metastatic 4T1 tumor cells were transduced to overexpress the pro-lymphangiogenic growth factor VEGF-C to model tumor lymphangiogensis in vivo. We confirmed that VEGF-C overexpressing tumors were more lymphangiogenic than control ones, as assessed by flow cytometry and immunohistochemistry analyses. As previously reported, tumor VEGF-C signaling also increased the metastatic burden in the lungs. Crucially, lymphangiogenic tumors were infiltrated with increased levels of immune cells, including CD4 T cells and macrophages. Upon treatment with an agonist of the stimulator of interferon genes (STING) pathway, lymphangiogenic 4T1 tumors responded more favorably to the therapy compared to the control tumors, as indicated by a reduced tumor growth in the VEGF-C group. Furthermore, gene expression analysis of human triple-negative breast tumor samples from The Cancer Genome Atlas revealed positive correlations between VEGFC expression and CD4 T-cell and macrophage gene expressions. Taken together, these data suggest that in triple-negative breast cancer, similarly to melanoma, VEGF-C signaling is beneficial to the primary tumor microenvironment upon combination with proper immunotherapy despite promoting metastasis in untreated tumors. Citation Format: Peyman Hosseinchi, Lambert Potin, Aslan Mansurov, Léa Maillat, Jeffrey A. Hubbell, Melody A. Swartz. The immunomodulatory roles of tumor-associated lymphatics in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B97.

Rationally Designed Polymer Conjugate for Tumor-Specific Amplification of Oxidative Stress and Boosting Antitumor Immunity.

The crosstalk between tumor and stroma cells is a central scenario in the tumor microenvironment (TME). While the predominant effect of tumor cells on immune cells is establishing an immunosuppressive context, tumor cell death at certain conditions will boost antitumor immunity. Herein, we report a rationally designed tumor specific enhanced oxidative stress polymer conjugate (TSEOP) for boosting antitumor immunity. The TSEOP is prepared by Passerini reaction between cinnamaldehyde (CA), 4-formylbenzeneboronic acid pinacol ester, and 5-isocyanopent-1-yne, followed by azide-alkyne click reaction with poly(l-glutamic acid)-graft-poly(ethylene glycol) monomethyl ether (PLG-g-mPEG). Under tumor stimuli condition, CA and quinone methide (QM) are quickly generated, which cooperatively induce strong oxidative stress, immunogenic tumor cell death (ICD), and activation of antigen presenting cells. In vivo studies show that the TSEOP treatment boosts tumor-specific antitumor immunity and eradicates both murine colorectal and breast tumors. This study should be inspirational for designing polymers as immunotherapeutics in cancer therapy.

CD5 blockade enhances ex vivo CD8+ Tcell activation and tumour cell cytotoxicity.

CD5 is expressed on Tcells and a subset of B cells (B1a). It can attenuate TCR signalling and impair CTL activation and is a therapeutic targetable tumour antigen expressed on leukemic T and B cells. However, the potential therapeutic effect of functionally blocking CD5 to increase Tcell anti-tumour activity against tumours (including solid tumours) has not been explored. CD5 knockout mice show increased anti-tumour immunity: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti-tumour response. Here, we show that ex vivo administration of a function-blocking anti-CD5 MAb to primary mouse CTLs of both tumour-naïve mice and mice bearing murine 4T1 breast tumour homografts enhanced their capacity to respond to activation by treatment with anti-CD3/anti-CD28 MAbs or 4T1 tumour cell lysates. Furthermore, it enhanced TCR signalling (ERK activation) and increased markers of Tcell activation, including proliferation, CD69 levels, IFN-γ production, apoptosis and Fas receptor and Fas ligand levels. Finally, CD5 function-blocking MAb treatment enhanced the capacity of CD8+ Tcells to kill 4T1-mouse tumour cells in an ex vivo assay. These data support the potential of blockade of CD5 function to enhance Tcell-mediated anti-tumour immunity.

Depleting tumor-associated Tregs via nanoparticle-mediated hyperthermia to enhance anti-CTLA-4 immunotherapy.

Aim: We aim to demonstrate that a local nanoparticle-mediated hyperthermia can effectively eliminate tumor-associated Tregs and thereby boost checkpoint blockade-based immunotherapy. Materials & methods: Photothermal therapy (PTT), mediated with systemically administered stealthy iron-oxide nanoparticles, was applied to treat BALB/c mice bearing 4T1 murine breast tumors. Flow cytometry was applied to evaluate both Treg and CD8+ T-cell population. Tumor growth following combination therapy of both PTT and anti-CTLA-4 was further evaluated. Results: Our data reveal that tumor-associated Tregs can be preferentially depleted via iron-oxide nanoparticles-mediated PTT. When combining PTT with anti-CTLA-4 immunotherapy, we demonstrate a significant inhibition of syngeneic 4T1 tumor growth. Conclusion: This study offers a novel strategy to overcome Treg-mediated immunosuppression and thereby to boost cancer immunotherapy.

Isothermal Microcalorimetry of Tumor Cells: Enhanced Thermogenesis by Metastatic Cells.

Tumor cells exhibit rewired metabolism. We carried out comparative analyses attempting to investigate whether metabolic reprograming could be measured by isothermal microcalorimetry. Intact metastatic cell lines of tongue cell carcinoma, human and murine melanoma, lung, and breast tumors consistently released more heat than non-metastatic cells or cells displaying lower metastatic potential. In tongue squamous carcinoma cells mitochondrial enriched extract reproduced the heat release pattern of intact cells. Cytochalasin D, an actin filament inhibitor, and suppression of metastasis marker Melanoma associated gene 10 (MAGEA10) decreased heat release. Uncoupling protein 2 was highly expressed in metastatic cells, but not in non-metastatic cells. Carnitine palmitoyl transferase-1 inhibitor, Etomoxir strongly inhibited heat release by metastatic cells, thus linking lipid metabolism to thermogenesis. We propose that heat release may be a quantifiable trait of the metastatic process.

Abstract C118: Transcriptome comparison of orthotopic and subcutaneous patient-derived xenografts

Abstract Background: Patient-derived xenograft (PDX) models are used for in vivo evaluation of oncology drugs. Tumors from cancer patients are implanted into immunodeficient mice either subcutaneously or orthotopically. Subcutaneous PDX models are more prevalently used because of easy operation and monitoring, albeit increasing evidence showed that the immune microenvironment in subcutaneous tissue is different from the original tract and is therefore less patient-like. By contrast, orthotopic PDX models have the capability to metastasize and match that of the donor patient well, but resulting in extensive inflammation. Previous studies showed that tumor location may affect immune response or result in different behavior of tumor expansion in mouse models [1-4]. There is yet insufficient study on the transcriptomic difference between subcutaneous and orthotopic PDX models. Method: Tumors for 10 PDX models—including four breast, four colorectal, and two liver cancers—were used to establish pairs of subcutaneous and orthotopic models. Tumors were dissected when reaching about 1000mm3, and subjected to transcriptomic profiling by RNAseq for gene expression comparison. Results: Using traditional alignment-based RNAseq analysis, we found hundreds of genes with different expression levels between subcutaneous and orthotopic models for the three cancers. Gene ontology analysis revealed that these differentially expressed genes are highly enriched in protein translation related biological processes and pathways, such as maturation of SSU-rRNA, cap-dependent translation initiation, and peptide chain elongation. Conclusion: Subcutaneous and orthotopic PDX models differ in transcriptomes that may affect protein translation and warrant further proteomics investigations. Reference: 1, Zhang, Yong, et al. “Real–Time GFP Intravital Imaging of the Differences in Cellular and Angiogenic Behavior of Subcutaneous and Orthotopic Nude–Mouse Models of Human PC–3 Prostate Cancer.” Journal of cellular biochemistry 117.11 (2016): 2546-2551. 2, Zhao, Xianda, et al. “Tumor location impacts immune response in mouse models of colon cancer.” Oncotarget 8.33 (2017): 54775. 3, Zhang, Yi, et al. “Establishment of a murine breast tumor model by subcutaneous or orthotopic implantation.” Oncology letters 15.5 (2018): 6233-6240. 4, Nakano, Kiyotaka, et al. “Difference in morphology and interactome profiles between orthotopic and subcutaneous gastric cancer xenograft models.” Journal of toxicologic pathology 31.4 (2018): 293-300. Citation Format: xiaoqian jiang, henry li, sheng guo. Transcriptome comparison of orthotopic and subcutaneous patient-derived xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C118. doi:10.1158/1535-7163.TARG-19-C118

Histone deacetylase inhibition promotes intratumoral CD8+ T-cell responses, sensitizing murine breast tumors to anti-PD1

Histone deacetylase (HDAC) inhibitors impair tumor cell proliferation and alter gene expression. However, the impact of these changes on anti-tumor immunity is poorly understood. Here, we showed that the class I HDAC inhibitor, entinostat (ENT), promoted the expression of immune-modulatory molecules, including MHCII, costimulatory ligands, and chemokines on murine breast tumor cells in vitro and in vivo. ENT also impaired tumor growth in vivo-an effect that was dependent on both CD8+ T cells and IFNγ. Moreover, ENT promoted intratumoral T-cell clonal expansion and enhanced their functional activity. Importantly, ENT sensitized normally unresponsive tumors to the effects of PD1 blockade, predominantly through increases in T-cell proliferation. Our findings suggest that class I HDAC inhibitors impair tumor growth by enhancing the proliferative and functional capacity of CD8+ T cells and by sensitizing tumor cells to T-cell recognition.

N-Dihydrogalactochitosan Potentiates the Radiosensitivity of Liver Metastatic Tumor Cells Originated from Murine Breast Tumors.

Radiation is a widely used therapeutic method for treating breast cancer. N-dihydrogalactochitosan (GC), a biocompatible immunostimulant, is known to enhance the effects of various treatment modalities in different tumor types. However, whether GC can enhance the radiosensitivity of cancer cells remains to be explored. In this study, triple-negative murine 4T1 breast cancer cells transduced with multi-reporter genes were implanted in immunocompetent Balb/C mice to track, dissect, and identify liver-metastatic 4T1 cells. These cells expressed cancer stem cell (CSC) -related characteristics, including the ability to form spheroids, the expression of the CD44 marker, and the increase of protein stability. We then ex vivo investigated the potential effect of GC on the radiosensitivity of the liver-metastatic 4T1 breast cancer cells and compared the results to those of parental 4T1 cells subjected to the same treatment. The cells were irradiated with increased doses of X-rays with or without GC treatment. Colony formation assays were then performed to determine the survival fractions and radiosensitivity of these cells. We found that GC preferably increased the radiosensitivity of liver-metastatic 4T1 breast cancer cells rather than that of the parental cells. Additionally, the single-cell DNA electrophoresis assay (SCDEA) and γ-H2AX foci assay were performed to assess the level of double-stranded DNA breaks (DSBs). Compared to the parental cells, DNA damage was significantly increased in liver-metastatic 4T1 cells after they were treated with GC plus radiation. Further studies on apoptosis showed that this combination treatment increased the sub-G1 population of cells, but not caspase-3 cleavage, in liver-metastatic breast cancer cells. Taken together, the current data suggest that the synergistic effects of GC and irradiation might be used to enhance the efficacy of radiotherapy in treating metastatic tumors.