Tumor-associated macrophages (TAMs) are the most abundant immune cells in primary solid tumors, including breast cancer, and typically exhibit an M2-like, immunosuppressive phenotype that promotes tumor growth. Given that TAMs can be repolarized through cytokine signaling, we propose a localized cytokine delivery depot using an injectable alginate cryogel to reprogram TAMs and create an inflammatory, anti-tumor TME. The cryogels were fabricated using cryogelation to generate a macroporous structure, followed by ionic crosslinking to enhance mechanical integrity while preserving pore size distribution. In vitro studies were conducted using bone marrow-derived macrophages, tumor-associated macrophages, and tumor explants. In vivo studies were conducted by orthotopically implanting breast tumors in the fat pads of FVB mice. Cell makeup and tissue composition were analyzed using qRT-PCR, flow cytometry, and Luminex panels. Statistical significance was determined using ANOVA and t-tests. In vitro, cryogels released chemokines and cytokines, attracted M2 macrophages, and repolarized them toward M1-like activities. In vivo, cryogel treatment increased the presence of M1 macrophages relative to M2 macrophages in both the primary tumor and lungs, reduced primary tumor growth, and decreased T-cell exhaustion. A localized, injectable cryogel depot successfully induces an inflammatory TME, leading to reduced tumor burden and T-cell exhaustion while avoiding systemic toxicities associated with cytokine delivery.

Abstract Introduction: We have reported that Class I/II HDACi sensitize PARPi resistant tumors, creating a possibility to expand the use of PARPi beyond homologous recombination deficient tumors. However, the underlying mechanism is still unknown. Here, we report leads obtained from differential gene expression in ovarian cancer cells treated with entinostat and olaparib. Methods: OVCAR3 cells were treated with 0.5 µM entinostat and 10 µM olaparib (alone and combined) for 6h, followed by RNA Seq. Cell cycle was analyzed by flow cytometry in SKOV-3 cells treated with olaparib and entinostat (alone and combined) for 24h, 48h and 72h. Results: RNA-Seq analysis showed reduced expression of genes that regulate G2/M checkpoint progression with both entinostat and olaparib. This correlated with flow cytometry results in SKOV-3 cells, where we observed accumulation of G2/M phase cells when treated with entinostat and olaparib. At 24h post treatment, olaparib and entinostat treated cells had higher population of cells in G2/M phase compared to control (18.5% and 16.7% respectively compared to 14.7% in control). Olaparib combined with entinostat further increased G2/M population to 21.7%. At 48h olaparib treated cells had 17.4% cells in G2/M compared to 14% in control. Entinostat treated cells had 18% G2/M cells. Combination treated cells had 19.7% G2/M cells. At 72h, olaparib and control had similar number of G2/M cells (9.2% and 9.7% respectively). Entinostat treated cells had 12% G2/M cells and combination treated cells had 13.6% cells at G2/M cells. This indicates that entinostat and olaparib disrupt progression of cells from G2/M phase, potentially leading to cell death before they can enter mitosis/cell division. Cell cycle analysis in OVCAR3 cells is in progress. Conclusion: Entinostat and olaparib cause G2/M cell cycle arrest in ovarian cancer cells. Entinostat maintains G2/M cell cycle arrest longer than olaparib, potentially extending the window of efficacy of the combination therapy. Validation of G2/M markers and other gene targets/pathways is in progress. Citation Format: Vijayalaxmi G. Gupta, Heng Liu, Rugang Zhang, Michael Onken, Andrew Wilson, Fiona Yull, Marta Crispens, Mary Mullen, Dineo Khabele. Entinostat alters cell cycle in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB421.

Abstract Background It has been shown that tumor microenvironment (TME) hydroxyapatite (HAP) is typically associated with many malignancies and plays a role in tumor progression and growth. Additionally, acidosis in the TME has been reported to play a key role in selecting for a more aggressive tumor phenotype, drug resistance and desensitization to immunotherapy for many types of cancers. TME‐HAP is an attractive target for tumor detection and treatment development since HAP is generally absent from normal soft tissue. We provide strong evidence that dissolution of hydroxyapatite (HAP) within the tumor microenvironment (TME‐HAP) using a novel therapeutic can be used to kill cancer cells both in vitro and in vivo with minimal adverse effects. Methods We developed an injectable cation exchange nano particulate sulfonated polystyrene solution (NSPS) that we engineered to dissolve TME‐HAP, inducing localized acute alkalosis and inhibition of tumor growth and glucose metabolism. This was evaluated in cell culture using 4T1, MDA‐MB‐231 triple negative breast cancer cells, MCF10 normal breast cells, and H292 lung cancer cells, and in vivo using orthotopic mouse models of cancer that contained detectable microenvironment HAP including breast (MMTV‐Neu, 4T1, and MDA‐MB‐231), prostate (PC3) and colon (HCA7) cancer using 18F‐NaF for HAP and 18F‐FDG for glucose metabolism with PET imaging. On the other hand, H292 lung tumor cells that lacked detectable microenvironment HAP and MCF10a normal breast cells that do not produce HAP served as negative controls. Tumor microenvironment pH levels following injection of NSPS were evaluated via Chemical Exchange Saturation (CEST) MRI and via ex vivo methods. Results Within 24 h of adding the small concentration of 1X of NSPS (~7 μM), we observed significant tumor cell death (~ 10%, p < 0.05) in 4T1 and MDA‐MB‐231 cell cultures that contain HAP but ⟨2% in H292 and MCF10a cells that lack detectable HAP and in controls. Using CEST MRI, we found extracellular pH (pHe) in the 4T1 breast tumors, located in the mammary fat pad, to increase by nearly 10% from baseline before gradually receding back to baseline during the first hour post NSPS administration. in the tumors that contained TME‐HAP in mouse models, MMTV‐Neu, 4T1, and MDA‐MB‐231, PC3, and HCA7, there was a significant reduction (p<0.05) in 18F‐Na Fuptake post NSPS treatment as expected; 18F‐ uptake in the tumor = 3.8 ± 0.5 %ID/g (percent of the injected dose per gram) at baseline compared to 1.8 ±0.5 %ID/g following one‐time treatment with 100 mg/kg NSPS. Of similar importance, is that 18F‐FDG uptake in the tumors was reduced by more than 75% compared to baseline within 24 h of treatment with one‐time NSPS which persisted for at least one week. Additionally, tumor growth was significantly slower (p < 0.05) in the mice treated with one‐time NSPS. Toxicity showed no evidence of any adverse effects, a finding attributed to the absence of HAP in normal soft tissue and to our therapeutic NSPS having limited penetration to access HAP within skeletal bone. Conclusion Dissolution of TME‐HAP using our novel NSPS has the potential to provide a new treatment paradigm to enhance the management of cancer patients with poor prognosis.

Abstract Poly ADP ribose inhibitors (PARPi) are most effective in ovarian cancer tumors with homologous recombination (HR) deficiency. Our group has shown that histone deacetylase inhibitors (HDACi) sensitize HR proficient ovarian cancer cells to PARPi. Our current efforts are directed towards understanding how this therapeutic regimen alters tumor-associated macrophages (TAMs) in murine models of high grade serous ovarian cancer. To investigate the effects of Ola combined with Ent, we used HR-proficient ID8 P53 wild-type and ID8 P53−/− syngeneic murine models. Mice were randomized into 4 groups: control, Ola, Ent and Ola+Ent. Mice (Ent and Ola+Ent) were pre-treated with 15 mg/kg entinostat or vehicle (Ola and Ola+Ent) for one week via oral gavage and then treated with vehicle/Ent/Ola (100 mg/kg)/Ola+Ent for two weeks. Mice were sacrificed 24 h after the last dose to harvest tumors and ascites. Tumors were processed for histology to determine cell proliferation (Ki67) and immune cell markers (CCL2, M1 and M2-like macrophages). Ascites fluid was processed for flow cytometric analysis of immune cells. Tumors from parental ID8 P53 wild type mice showed significantly lower cell proliferation marker Ki67 (P<0.05), higher anti-tumorigenic M1-like CCL2 (P<0.05), and lower pro-tumorigenic M2-like mannose receptor (P<0.05) in Ent and Ola+Ent groups compared to vehicle and Ola. Ascites showed no significant change in anti-tumorigenic M1-like macrophages, but significantly increased pro-tumorigenic M2-like macrophages (P<0.005). Arginase-1, another marker of M1 macrophage, assessed by IF also confirmed significant decrease in pro-tumorigenic M2-like macrophage in Ent and Ola+Ent treated mice tumors. Additional immune cell markers identified in ascites are being analyzed. To summarize, in ID8 syngeneic mouse models, Ola and Ent treatment exerted anti-tumorigenic effects in tumors but potentially pro-tumorigenic effects in ascites. In conclusion, concomitant targeting of tumor TAMs and ascites TAMs may be a therapeutic regimen to investigate in the future. Citation Format: Vijayalaxmi Gupta, Katherine Roby, SImona Miceska, Andrew Wilson, Fiona Yull, Marta Crispens, Wendy Zhang, Dineo Khabele. Entinostat alters the M1/M2 ratio in ascites compared to tumors derived from an ID8 murine model of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A100.

Tumor-associated macrophages (TAMs) represent more than 50% of the immune cells in breast tumors, even in immune dysregulated, ‘cold’ tumors. TAMs that express an M2-like phenotype promote tumor growth and create an immunosuppressive tumor microenvironment (TME). High levels of M2-like TAMs correlate with a poor prognosis. However, macrophages display phenotypic plasticity and can be repolarized from one phenotype to another through signaling molecules, such as cytokines. Macrophages that express an M1-like phenotype are inflammatory and can induce anti-tumor immunity. Administration of pro-inflammatory cytokines reprogram M2 TAMs toward an M1-like phenotype that can reduce tumor growth and stimulate an inflammatory response within the TME. However, systemic delivery of these inflammatory cytokines generates toxic, dose-limiting off target effects. This work seeks to develop a localized delivery approach that enables TAM polarization toward M1 programs while abrogating systemic toxicities. We have developed an injectable alginate cryogel (hydrogel fabricated at -20oC) to act as localized delivery depot for inflammatory cytokines and macrophage-specific chemokines to repolarize TAMs. To delay the release of our chemoattractant relative to our repolarizing cytokines, we are developing a second generation cryogel, loaded with free chemokines and poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulated with cytokines to delay their release. With our first generation cryogel (without nanoparticles), we used qRT-PCR, flow cytometry, Luminex panels, and single cell RNAseq to characterize the impact of treatment. For in vitro studies, bone marrow derived macrophages were extracted from FVB mice, while TAMs and tumor explants were extracted from the spontaneous mammary tumors of PyMT mice. It was found that our cryogel significantly increased M1 markers and decreased M2 markers in all three in vitro models.Peritumoral injection of the cryogel system into FVB female mice with orthotopic mammary tumors resulted in significantly suppressed tumor growth, an increase in T cell infiltration, and an increase in the M1:M2 ratio of TAMs. This activated immune response and elevated presence of T cells suggests that localized chemokine/cytokine treatment ‘primes’ the TME, potentially making it more susceptible to immunotherapies that rely on T cells, such as immune checkpoint blockade.We have concluded that our first-generation gel demonstrates the potential of priming the TME to slow tumor growth. Characterization of our second generation, nano-in-cryogel system is currently underway. Based on mathematical modeling, we hypothesize that the nano-in-cryogel will induce a more pronounced repolarization effect by allowing the chemoattractant to induce M2 TAM migration prior to the release of the inflammatory, reprogramming cytokines from the PLGA nanoparticles. Citation Format: Sydney Ros Henriques, Ori Z. Chalom, Evan B. Glass, Sohini Roy, Abigail E. Manning, Benjamin C. Hacker, Marjan Rafat, Laura C. Kennedy, Fiona E. Yull, Young J. Kim, Todd D. Giorgio. Tumor-associated macrophage reprogramming by novel nano-in-cryogel biomaterials for breast cancer immunotherapy [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 2719.

Supplementary Figure 1 from The Nuclear Factor-κB Pathway Controls the Progression of Prostate Cancer to Androgen-Independent Growth

Supplementary Figure S8 - PDF file 1084K, Figure S8: Effects of SSTC-104 on Wnt-regulated tissues

Background: Ovarian cancer is the most lethal gynecologic malignancy, with a 5-year survival rate of less than 50%. To have a major impact on improving clinical outcomes, a better understanding of the progression of disease and identification of truly novel treatment strategies is critical. Canonical nuclear factor-kappaB (NF-κB) signaling has been extensively studied in preclinical ovarian cancer models; however, systemic NF-κB inhibitors have had disappointing results in early clinical trials. Whether the alternative, non-canonical NF-κB signaling pathway (nc-NF-κB) represents a distinct and improved target for therapy in ovarian cancer is unknown. Here, we aim to investigate the role played by nc-NF-κB signaling both in malignant epithelial cells and in macrophages, a key immune cell component in the tumor microenvironment. Repolarizing tumor-associated macrophages (TAMs) away from pro-tumor M2-like functions towards an anti-tumor M1-like phenotype is a critical priority in macrophage-directed therapies. Methods: In ovarian cancer cell lines, mouse ovarian TAMs cultured ex vivo, and immortalized bone marrow-derived macrophages (BMDMs), nuclear levels of p52 were measured by western blot as a read-out of active nc-NF-κB signaling. Effects of macrophage polarization on nc-NF-κB signaling were determined in unpolarized (M0), M1 polarized (LPS/IFNγ treated) or M2 polarized (IL-4 treated) BMDMs. Finally, effects of inhibiting nc-NF-κB using a specific peptide inhibitor of p52 nuclear import (SN52) on cancer cell growth and macrophage phenotype were determined in sulforhodamine B growth assays and by western blot analysis of PCNA (proliferation marker) and arginase-1 (M2 macrophage marker). Results: We observed high levels of p52 expression in the majority of ovarian cancer cell lines, and in our cultured macrophage models. The functional relevance of these observations was confirmed by experiments showing reduced growth in ovarian cancer cells treated with SN52 and reduced expression of arginase-1 in SN52-treated macrophages. We saw similar results in breast cancer cells and mouse mammary TAMs, showing that nc-NF-κB signaling may also have pro-tumor effects in other cancer types. Complementary studies showed that M2 polarized macrophages expressed markedly higher levels of nuclear p52 than observed in M0 or M1 cells. Based on these promising results, experiments (i) using transgenic mice to allow targeted, inducible activation of nc-NF-κB in macrophages during ovarian cancer progression and (ii) testing the therapeutic effects of SN52 in these cancer models are underway. Conclusions: Although further studies are required to establish the translational relevance of our findings, we hope to identify non-canonical NF-κB signaling as a new therapeutic target in malignant epithelium and macrophages in ovarian cancer and potentially other cancer types. Citation Format: Andrew J. Wilson, Dominique Parker, Robert Cheng, Todd Giorgio, Marta Crispens, Fiona Yull. Investigating the role of non-canonical NF-kappaB signaling in tumor cells and macrophages in ovarian 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 4973.

Figure S2. Deficiency in IL-5 does not affect infiltration of lungs with T and B lymphocytes and NK cells during initial stages of metastatic load of LLC cells.

<p>Figure S1: Myeloid Ikkβ is required for anti-tumorigenic immunity. Figure S2. Myeloid cell depletion alters tumor immunity. Figure S3. Ikkβ loss alters macrophage phagocytosis capacity. Figure S4. Myeloid Ikkβ deletion reduced the cytotoxicity of T cells Figure S5. Cytokines play a major role in orchestrating the cellular make-up of the tumor microenvironment.</p>

Poly ADP ribose inhibitors (PARPi) are most effective in ovarian cancer tumors with homologous recombination (HR) deficiency. Our group has shown that histone deacetylase inhibitors (HDACi) sensitize HR proficient ovarian cancer cells to PARPi. Our current efforts are directed towards understanding how this therapeutic regimen alters tumor-associated macrophages (TAMs) in murine models of high grade serous ovarian cancer. To investigate the effects of Ola combined with Ent, we used HR-proficient ID8 P53 wild-type and ID8 P53−/− syngeneic murine models. Mice were randomized into 4 groups: control, Ola, Ent and Ola+Ent. Mice (Ent and Ola+Ent) were pre-treated with 15 mg/kg entinostat or vehicle (Ola and Ola+Ent) for one week via oral gavage and then treated with vehicle/Ent/Ola (100 mg/kg)/Ola+Ent for two weeks. Mice were sacrificed 24 h after the last dose to harvest tumors and ascites. Tumors were processed for histology to determine cell proliferation (Ki67) and immune cell markers (CCL2, M1 and M2-like macrophages). Ascites fluid was processed for flow cytometric analysis of immune cells. Tumors from parental ID8 P53 wild type mice showed significantly lower cell proliferation marker Ki67 (P<0.05), higher anti-tumorigenic M1-like CCL2 (P<0.05), and lower pro-tumorigenic M2-like mannose receptor (P<0.05) in Ent and Ola+Ent groups compared to vehicle and Ola. Ascites showed no significant change in anti-tumorigenic M1-like macrophages, but significantly increased pro-tumorigenic M2-like macrophages (P<0.005). ID8 P53−/− mice tumors showed significantly lower Ki-67 (P<0.05) in Ola+Ent group compared to vehicle and mono-treatments. Ascites showed no significant change in total macrophage or pro-tumorigenic M2-like macrophages, but a significant decrease in anti-tumorigenic M1-like macrophages in Ent and Ola+Ent groups compared to vehicle. To summarize, in HR proficient ID8 P53 wildtype and ID8 P53−/− syngeneic mouse models, Ola and Ent treatment exerted anti-tumorigenic effects in tumors but potentially pro-tumorigenic effects in ascites. In conclusion, concomitant targeting of tumor TAMs and ascites TAMs may be a therapeutic regimen to investigate in the future. Citation Format: Vijayalaxmi G. Gupta, Tyler Woodard, Simona Miceska, Bisiyao Fashemi, Sangappa Chadchan, Wendy Zhang, Katherine Roby, Andrew Wilson, Fiona Yull, Marta Crispens, Sumanta Naik, Asya Smimov, Christina Stallings, Dineo Khabele. Olaparib combined with entinostat exerts differential effects on tumor-associated macrophages in tumors compared to ascites in syngeneic HR-proficient murine models of ovarian 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 3654.

Figure S1. Lung eosinophils (Eos), identified as CD45+/CD11c-/ F4/80+/siglec-F+ cells using flow cytometry are A) negative for CD68, B) express high levels of CCR3, and C) express low levels of Gr1 compared to the CD45+/Gr1hi neutrophils (PMN).

Supplementary Figure S10 4882K, Figure S10a and S10b: Nuclear beta-catenin accumulation in human synovial sarcoma tumors

Supplementary Methods and Legends - PDF file 130K, Methods:Additional information on experimental techniques Legends: describing supplementary figures

PDF file - 453KB, Supplementary Figure 1. NF-kappaB signaling is continuously activated in the prostate of IkappaBalpha+/- mouse. Supplementary Figure 2. NF-kappaB signaling activated in the prostate of Myc/IkappaBalpha bigenic mouse. Supplementary Figure 3. Continuous activation of NF-kappaB signaling promotes PCa progression in the Hi-Myc transgenic mouse. Supplementary Figure 4. The NF24 gene signature predicts significant differences in the overall cancer-specific survival of the PCa patients. Supplemental Figure 5. Molecular network analysis using Ingenuity Pathway Analysis (IPA). Supplemental Figure 6. NF-kappaB signaling is activated/inactivated in PCa cells by infecting with IKK2-EE or IKK2-KD retroviral vectors. Supplemental Figure 7. Activation of NF-kappaB signaling increases JNK phosphorylation in PCa cells. Supplemental Figure 8. Blocking JNK signaling inhibits NF-kappaB induced invasive ability efficiently in PCa cells. Tables 1 and 2. Matched human homolog genes. Supplementary Table 3. Stratification of 77 PCa patients.

Kirsten rat sarcoma virus (KRAS)-mutant cancers are frequent, metastatic, lethal, and largely undruggable. While interleukin (IL)-1β and nuclear factor (NF)-κB inhibition hold promise against cancer, untargeted treatments are not effective. Here, we show that human KRAS-mutant cancers are addicted to IL-1β via inflammatory versican signaling to macrophage inhibitor of NF-κB kinase (IKK) β. Human pan-cancer and experimental NF-κB reporter, transcriptome, and proteome screens reveal that KRAS-mutant tumors trigger macrophage IKKβ activation and IL-1β release via secretory versican. Tumor-specific versican silencing and macrophage-restricted IKKβ deletion prevents myeloid NF-κB activation and metastasis. Versican and IKKβ are mutually addicted and/or overexpressed in human cancers and possess diagnostic and prognostic power. Non-oncogene KRAS/IL-1β addiction is abolished by IL-1β and TLR1/2 inhibition, indicating cardinal and actionable roles for versican and IKKβ in metastasis.

Supplementary Figure S9 - PDF file 867K,Figure S9: The SYT-SSX2 domain SSXRD executes -catenin activation and myogenic differentiation

Supplementary Figures S2 and S3 - PDF file 2332K, Figure S2:SG3 tumors are positive for beta-catenin and Myf5 Figure S3:Normal development of Myf5 myoblasts in beta-catenin knockout mice