CDK4/6 inhibitors are standard-of-care for metastatic estrogen receptor-positive (ER+) breast cancer, yet the development of resistance remains a significant clinical hurdle. While CDK4/6 inhibitors are primarily recognized for their ability to induce cytostasis, their role in modulating innate immune responses remains poorly defined. Here, we demonstrated that CDK4/6i treatment remodels the tumor cell surface to favor recognition and elimination by Natural Killer (NK) cells. Using a diverse biobank of patient-derived organoids (PDOs), we found that CDK4/6 inhibition robustly upregulated the adhesion molecule ICAM-1 and the NKG2D stress ligands (ULBP2/5/6 and MICA/B). This NK-engaging cell surface phenotype was driven by a bifurcated signaling network: NF-κB signaling orchestrated ICAM-1 induction, while the PI3K/mTOR pathway regulated the expression of stress ligands. Functional assays confirmed that these ligands were indispensable for NK cell-mediated elimination of breast cancer cells. In vivo studies using ER+ PDX models revealed that a brief seven-day primer treatment with the CDK4/6 inhibitor abemaciclib was sufficient to sensitize tumors to NK cell therapy, significantly inhibiting tumor growth and prolonging survival. We also observed efficacy with a concurrent dosing strategy that delayed the onset of acquired resistance. These findings provide a mechanistic rationale for combining CDK4/6 inhibitors with NK cell therapy. This "prime and kill" approach offers a promising strategy to overcome therapeutic resistance and improve outcomes for patients with metastatic ER+ breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive malignancy with limited therapeutic options, highlighting the urgent need to define tumor-intrinsic drivers of progression. Here, we uncover a previously unrecognized Psoriasin (S100A7)-driven signaling axis that promotes TNBC growth and identifies cytosolic phospholipase A2 (cPLA2) as a therapeutically actionable vulnerability. Mechanistically, S100A7 engages the RAGE receptor to activate NF-κB, inducing cPLA2, which subsequently upregulates NHERF2 and suppresses GSK3β, sustaining oncogenic signaling that drives proliferation, survival, and metastasis. Disruption of this S100A7→cPLA2→NHERF2 cascade restores GSK3β activity and markedly attenuates tumorigenicity. Genetic ablation of S100A7 in knockout mice or knockdown of NHERF2 suppresses tumor burden, whereas pharmacologic inhibition of GSK3β restores tumor progression, revealing the hierarchical organization and functional importance of this pathway. Using complementary preclinical models, including mammary gland-specific S100A7-overexpressing mice, patient-derived organoids (PDOs), and xenografts (PDXs), we show that targeting this axis, genetically or pharmacologically, effectively suppresses tumor growth and spontaneous metastasis. Notably, combining a cPLA2 inhibitor with our developed first-in-class S100A7-neutralizing antibody produces synergistic anti-tumor effects. Clinically, co-elevation of S100A7 with cPLA2 or NHERF2 associates with poor recurrence-free and distant metastasis-free survival, highlighting the translational relevance of this tumor-intrinsic signaling axis. Collectively, these findings provide both conceptual and translational advances by defining a novel signaling cascade underlying TNBC aggressiveness, revealing an actionable tumor-intrinsic vulnerability, and establishing a rationale for combinatorial interventions that may extend to other cancers with similar intrinsic dependencies.

Melanoma is the deadliest form of skin cancer and still has a poor prognosis when in metastatic stage. Currently available treatments often lead to resistance development and several adverse effects. Plant-derived polysaccharides have demonstrated diverse immunological and antitumor activities. This study explored the immunomodulatory and antimelanoma potential of an homogalacturonan from passion fruit peel (HG-PFP). HG-PFP induced a pro-inflammatory phenotype in macrophages, enhancing nitric oxide, reactive oxygen species, and cytokine release. In B16-F10 melanoma-bearing mice, HG-PFP (50 mg/kg) reduced tumor growth by 60% and lung colonization by 54%, with no adverse effects. Even though the tumor microenvironment macrophage population was not altered by the treatment, monocyte chemoattractant protein-1 (MCP-1) levels were changed in both subcutaneous tumor and lung colonization models. Considering melanoma high heterogeneity and subtypes and to further expand HG-PFP treatment effects investigation, we evaluated human preclinical melanoma model bearing melanoma driver mutations (BRAF, NRAS, and NF1), using patient-derived organoids (PDOs). HG-PFP treatment inhibited NF1 mutant PDOs growth by reducing cells viability and impaired organoids formation and growth in NRAS mutant samples, while showing no effects in BRAF mutant samples. Taking together these findings provide a preclinical perspective on therapeutic potential of HG-PFP across different melanoma subtypes.

BACKGROUND:Therapy-induced senescence (TIS) is a common outcome of diverse anticancer treatments, including chemotherapy, radiation, and small-molecule inhibitors. Senescence is characterized by stable growth arrest and the senescence-associated secretory phenotype (SASP), which includes various immune mediators. As the role of the immune system in controlling cancer becomes increasingly appreciated, understanding the impact of TIS on the tumor immune microenvironment (TIME) is critically important. Here, we investigated how senescence can be leveraged to enhance antitumor immune responses. METHODS:We investigated the effects of an Aurora kinase A inhibitor (AURKAi), a potent inducer of senescence in melanoma models, using transcriptome and secretome profiling. The role of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway was investigated using imaging, inhibitors, and gene knockout. We also examined the effect of AURKAi on the surface expression of major histocompatibility complex class I (MHC-I) and programmed death-ligand 1 (PD-L1), as well as on signal transducer and activator of transcription 1 (STAT1) activation. In vivo, the effects of AURKAi treatment on the TIME were investigated using spectral cytometry and cell depletion studies. Finally, we assessed combining AURKAi with immune checkpoint blockade (ICB), adoptive cell therapy, and natural killer (NK) cell therapy in murine models. RESULTS:We observed significant gene expression rewiring and the secretion of immune-related mediators and chemokines associated with AURKAi-induced senescence, which was favorably modulated by a senolytic BCL-2/xL inhibitor navitoclax. Mechanistically, AURKAi induced the formation of micronuclei linked with the activation of the cGAS-STING pathway, which, in turn, initiated pro-inflammatory transcriptional programs. Senescent melanoma cells exhibited increased surface expression of MHC-I and PD-L1, along with interferon regulatory factor 3 (IRF3) and STAT1 activation, indicating enhanced immunogenicity. In vivo, AURKAi treatment significantly enriched the TIME with activated CD8+ T cells and NK cells, with depletion studies confirming their critical role in antitumor effects. While combining AURKAi with immune checkpoint blockade was not beneficial, AURKAi pretreatment significantly augmented T and NK cell therapies in murine models, resulting in enhanced tumor control and prolonged survival. CONCLUSIONS:These findings suggest that senescence, specifically when induced by an AURKAi, can be harnessed to promote effector cell engagement in immune-cold tumors, thereby enhancing responsiveness to cell therapies.

Epidemiological studies associate an increase in breast cancer risk, particularly triple-negative breast cancer (TNBC), with lack of breastfeeding. This is more prevalent in African American women, with significantly lower rate of breastfeeding compared to Caucasian women. Prolonged breastfeeding leads to gradual involution (GI), whereas short-term or lack of breastfeeding leads to abrupt involution (AI) of the breast. Our previous study utilizing a murine model demonstrated precancerous changes, specifically hyperplasia, a non-obligate precursor of breast cancer in the mammary glands of AI mice. Here we investigated mechanisms during early events of AI that prompts precancerous changes in mouse mammary glands. Uniparous FVB/N mice were randomized to AI and GI on postpartum day 7 when all pups were removed from AI dams. GI dams were allowed to nurse the pups till day 31. Cell death kinetics and gene expression were assessed by TUNEL assay and qPCR respectively. Immune cell changes were investigated by flow cytometry, cytokine array and multiplex immunofluorescence. 3D-organoid cultures were used for in vitro assay of luminal progenitor cells. AI results in rapid cell death, DNA repair response, and immunosuppressive myeloid cells infiltration, leading to a chronically inflamed microenvironment. GI elicits a more controlled immune response and extended cell death. At the peak of cell death, AI glands harbored more immunosuppressive myeloid-derived suppressor cells (MDSCs) and CD206 + M2-like macrophages, known to promote oncogenic events, compared to GI glands. AI glands exhibit an enrichment of CCL9-producing MDSCs and CD206 + M2-like macrophages that promote expansion of ELF5 + /ERα- luminal cells, both in vitro and in vivo. Multiplex imaging of AI glands demonstrated an increase in ELF5 + /WNT5a + luminal cells alongside a reduction in the ELF5 + /ERα + population when involution appeared histologically complete. A significantly higher number of CD206 + cells in post involution AI gland attests to a chronically inflamed state induced by AI. Our findings reveal significant disparities between AI and GI gland dynamics at the early phase of involution. CCL9, secreted by immune cells at the peak of cell death promotes expansion of Elf5 + /ERα- luminal progenitor cells, the putative precursors of TNBC connecting early events of AI with increased breast cancer risk.

Real-time imaging of multiple immunomarkers in vivo is crucial to predict treatment response and to understand the complex interplay of immune cells such as CD8+ T cells and natural killer (NK) cells in the tumor microenvironment (TME). Since dynamic changes occur in these immune cells during combinatorial immunotherapies, these changes cannot be captured by single biomarker tracking or by ex vivo measurement of receptors at the endpoint of treatment. There is an unmet need for imaging probes that cohesively integrate the merits of multiple imaging modalities while surmounting the challenges of each modality. Here, we have developed multimodal gold nanostars (MGNs) functionalized with Raman reporters, immune-targeting antibodies, and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-chelated 64Cu radiolabels. These MGNs integrate the high sensitivity and whole-body imaging of immunoPET (positron emission tomography) with high spatial resolution and multiplexing of Raman spectroscopy for real-time in vivo detection of recruited CD8+ T cells and NKp46+ NK cells in the TME. MGNs predict treatment response to antiPD-L1 + antiCD47 combination immunotherapies in both immunologically "hot" CT26 murine tumors that are responders of therapy and immunologically "cold" 4T1 murine tumors that are poor responders. In vivo endpoints were validated with conventional assays (immunofluorescence, ELISA, and flow cytometry) to confirm immune cell recruitment. Our results show that MGNs enable imaging capabilities that cannot be achieved with either modality alone, allowing early detection of immunomarkers to improve therapeutic outcomes for responders and accelerate clinical decisions for poor responders.

Abstract Breast cancer is the most common cancer diagnosis worldwide, and an unfortunately high number of women die every year due to a lack of effective therapeutic strategies for metastatic breast cancer. Patients with brain metastases in particular have a significantly poorer prognosis than women with metastasis to other sites (e.g., lung, liver, bone). Previous studies by our group identified a ligand-receptor complex in the tumor microenvironment (TME) that promotes metastasis of breast cancer to the brain. This interaction between platelet-derived growth factor-BB (PDGFB), produced by cancerous breast epithelial cells, and its receptor, platelet-derived growth factor receptor beta (PDGFRβ), expressed by mesenchymal cells, is also prognostic of breast cancer metastasis to the brain in patients. The mechanism of how the PDGFB-PDGFRβ pathway mediates brain metastasis is still unclear and is the focus of our current work. To further analyze the biological effects of PDGFB, our group evaluated gene expression changes between primary human breast tumors expressing high PDGFB versus those expressing low PDGFB and found significantly more immune signaling in the low-expressing tumors. These results led us to hypothesize that the PDGFB-PDGFRβ pathway creates an immunosuppressive microenvironment, allowing for increased metastasis of cancerous cells. To test this, flow cytometry was completed on the brains, spleens, lungs, and livers of control mice and mutant mice expressing stromal-specific hyperactive PDGFRβ following intracardiac injection of PDGFB-expressing mammary tumor cells. No significant changes in immune cell populations were observed in non-tumor-bearing mice. However, in the brains of tumor-bearing mutant mice, there are significant changes to the myeloid lineage. Specifically, analysis at day 10 post-injection (prior to detectable metastatic lesions) revealed diminished CD45+CD11b+ populations. Segregating by CD45hi/CD45lo confirmed a profound decrease in CD45loCD11b+F4/80+ microglia, likely a tumor-suppressive population. Looking at day 16 post-injection (detectable metastatic lesions), there is a shift towards an increase in CD45+ CD11b+GR1+ infiltrating myeloid-derived suppressor cells (MDSCs) in experimental mice, likely a tumor-promoting population. Minimal changes were observed in the lungs and livers at any time point. Confirmatory immunofluorescence staining showed a decrease in tumor-infiltrating microglia (Iba1+) and a significant increase in tumor-infiltrating peripheral macrophages (F4/80+), mirroring the flow data. This experiment was then repeated to test the contribution of the ligand (PDGFB), where a significant increase of infiltrating microglia (Iba1+) was observed upon tumor cell knockdown of PDGFB. Together, these results suggest that the PDGFB-PDGFRβ pathway creates an immunosuppressive environment in the brain TME at least in part through reorchestration of the myeloid populations. Citation Format: Alexis A Mossing, Johnathon G Schiebel, Rebecca L Packard, Amrendra Kumar, Sarah A Steck, Nathaniel S Grabinski, Katie A Thies, Bri Wasik, Tasneem Ariswala, Gary Tozbikian, Johnathan Godbout, Steven T Sizemore, Paul R Lockman, Anna E Vilgelm, Gina M Sizemore. Stromal PDGFRβ hyperactivation modulates the myeloid lineage in the brain metastatic microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A085.

BACKGROUND:Evidence suggests that COVID-19 predisposes to cardiovascular diseases (CVDs). While monocytes/macrophages play a central role in the immunopathogenesis of atherosclerosis, less is known about their immunopathogenic mechanisms that lead to CVDs during COVID-19. Natural killer (NK) cells, which play an intermediary role during pathologies like atherosclerosis, are dysregulated during COVID-19. Here, we sought to investigate altered immune cells and their associations with CVD risk during severe COVID-19.METHODS:We measured plasma biomarkers of CVDs and determined phenotypes of circulating immune subsets using spectral flow cytometry. We compared these between patients with severe COVID-19 (severe, n=31), those who recovered from severe COVID-19 (recovered, n=29), and SARS-CoV-2-uninfected controls (controls, n=17). In vivo observations were supported using in vitro assays to highlight possible mechanistic links between dysregulated immune subsets and biomarkers during and after COVID-19. We performed multidimensional analyses of published single-cell transcriptome data of monocytes and NK cells during severe COVID-19 to substantiate in vivo findings.RESULTS:During severe COVID-19, we observed alterations in cardiometabolic biomarkers including oxidized-low-density lipoprotein, which showed decreased levels in severe and recovered groups. Severe patients exhibited dysregulated monocyte subsets, including increased frequencies of proinflammatory intermediate monocytes (also observed in the recovered) and decreased nonclassical monocytes. All identified NK-cell subsets in the severe COVID-19 group displayed increased expression of activation and tissue-resident markers, such as CD69 (cluster of differentiation 69). We observed significant correlations between altered immune subsets and plasma oxidized-low-density lipoprotein levels. In vitro assays revealed increased uptake of oxidized-low-density lipoprotein into monocyte-derived macrophages in the presence of NK cells activated by plasma of patients with severe COVID-19. Transcriptome analyses confirmed enriched proinflammatory responses and lipid dysregulation associated with epigenetic modifications in monocytes and NK cells during severe COVID-19.CONCLUSIONS:Our study provides new insights into the involvement of monocytes and NK cells in the increased CVD risk observed during and after COVID-19.

TTK spindle assembly checkpoint kinase is an emerging cancer target. This preclinical study explored the antitumor mechanism of TTK inhibitor OSU13 to define a strategy for clinical development. We observed prominent antitumor activity of OSU13 in melanoma, colon and breast cancer cells, organoids derived from patients with melanoma, and mice bearing colon tumors associated with G2 cell cycle arrest, senescence, and apoptosis. OSU13-treated cells displayed DNA damage and micronuclei that triggered the cytosolic DNA-sensing cGAS/STING pathway. STING was required for the induction of several proteins involved in T cell recruitment and activity. Tumors from OSU13-treated mice showed an increased proportion of T and NK cells and evidence of PD-1/PD-L1 immune checkpoint activation. Combining a lowtoxicity dose of OSU13 with anti-PD-1 checkpoint blockade resulted in prominent STING- and CD8+ T cell-dependent tumor inhibition and improved survival. These findings provide a rationale for utilizing TTK inhibitors in combination with immunotherapy in STING-proficient tumors.

Abstract Introduction: A meta-analysis of 47 global epidemiological studies highlights a higher breast cancer risk in women who did not breastfeed or breastfed for a short time. Further studies showed this is especially true for triple-negative breast cancer (TNBC) patients. Premenopausal AA women (AAW) have a lower prevalence of breastfeeding and a higher incidence of TNBC and mortality. Our previous study compares short-term breastfeeding, abrupt involution (AI) with prolonged breastfeeding called gradual involution (GI), revealing that AI alone induces ductal hyperplasia four months postpartum. Our current investigation delves into early events during AI versus GI, employing a comprehensive approach encompassing histology, gene expression, and myeloid cell involvement. Methods: Utilizing FVB female mice, we conducted a comparative analysis of AI and GI. AI involved early pup removal, while GI was achieved through staggered weaning. The evaluation included analysis of histomorphology, gene/protein expression, and myeloid cell infiltration. Sequential mammary gland (MG) changes were monitored through H&E staining, TUNEL assay, and DNA damage analysis. 3D-organoid cultures of luminal progenitors (LPs) were employed to assess the impact of AI versus GI. qRT-PCR, IHC, Western blot, and flow cytometry/multiplex imaging were employed for the differential expression analysis of molecular and cellular factors associated with AI/GI. Results: Our research showed that AI had early adipocyte repopulation, rapid cell death, DNA repair, and myeloid cell infiltration, resulting in a chronically inflamed microenvironment. In contrast, the GI triggers a controlled immune response and prolonged cell death, facilitating comprehensive remodeling of the MG. Our flow cytometric or multiplexing imaging analyses revealed that AI-affected glands exhibit an enrichment of CCL9-producing CD206+ M2-like macrophages and CD11b+Gr1+ myeloid-derived suppressor cells. Moreover, exogenous CCL9 treatment on LPs in 3D-organoid culture results in disorganized acinar-type organoids, mirroring morphological differences observed in LPs from AI mammary glands on day56 PPM. Further analysis of CCL9 treated organoids revealed the expansion of Esr1- LPs population in ex-vivo organoid culture which might indicate an increase in the putative cells of origin of TNBC. Conclusion: Our studies comparing AI and GI demonstrate that AI is producing a pro-tumorigenic environment in the breast. It is important to note that prolonged breastfeeding protects the breast, although this cannot be a singular risk factor for TNBC. Therefore, understanding the mechanism will lead to prevention strategies to improve outcomes for all women but, has the potential to have a significant benefit in AAW. Citation Format: Sanjay Mishra, Neelam Shinde, Maria Cuitino, Morgan Bauer, Dinesh Ahirwar, Vijaya Bharti, Kate Ormiston, Resham Mawalkar, Sara Alsammerai, Gautam Sarathy, Xiaoli Zhang, Anna Vilgelm, Ramesh Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Understanding the link between breastfeeding and the risk of breast cancer through comparative analysis of the murine-based model of mammary gland involution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2138.

Head and neck squamous cell carcinoma (HNSCC) is a significant public health concern worldwide. Immunomodulatory targets in the HNSCC tumor microenvironment are crucial to enhance the efficacy of HNSCC immunotherapy. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that has been linked to poor prognosis in many cancers, but the mechanistic role of MIF in HNSCC remains unclear. Using a murine orthotopic oral cancer model in Mif + / + or Mif- /- mice, we determined the function of host derived MIF in HNSCC tumor development, metastasis as well as localized and systemic tumor immune responses. We observed that Mif- /- mice have decreased tumor growth and tumor burden compared to their wild-type counterparts. Flow cytometric analysis of immune populations within the primary tumor site revealed increased Th1 and cytotoxic T cell recruitment to the HNSCC tumor microenvironment. Within the tumors of Mif- /- mice, MIF deletion also enhanced the effector function of anti-tumoral effector CD8 + T cells as well as Th1 cells and decreased the accumulation of granulocytic myeloid derived suppressor cells (g-MDSCs) in the tumor microenvironment. Furthermore, MDSCs isolated from tumor bearing mice chemotactically respond to MIF in a dose dependent manner. Taken together, our results demonstrate a chemotactic and immunomodulatory role for host derived MIF in promoting HNSCC and suggest that MIF targeted immunomodulation is a promising approach for HNSCC treatment.

Supplementary Table 1 and Figures 1-7

Current methodologies for developing PDX in humanized mice in preclinical trials with immune-based therapies are limited by GVHD. Here, we compared two approaches for establishing PDX tumors in humanized mice: (1) PDX are first established in immune-deficient mice; or (2) PDX are initially established in humanized mice; then established PDX are transplanted to a larger cohort of humanized mice for preclinical trials. With the first approach, there was rapid wasting of PDX-bearing humanized mice with high levels of activated T cells in the circulation and organs, indicating immune-mediated toxicity. In contrast, with the second approach, toxicity was less of an issue and long-term human melanoma tumor growth and maintenance of human chimerism was achieved. Preclinical trials from the second approach revealed that rigosertib, but not anti-PD-1, increased CD8/CD4 T cell ratios in spleen and blood and inhibited PDX tumor growth. Resistance to anti-PD-1 was associated with PDX tumors established from tumors with limited CD8+ T cell content. Our findings suggest that it is essential to carefully manage immune editing by first establishing PDX tumors in humanized mice before expanding PDX tumors into a larger cohort of humanized mice to evaluate therapy response.

Supplementary figures S1-4. Fig. S1: Cells were treated with MLN8237 (1µM) for five days. Real-Time PCR was performed for senescence markers as indicated. Fig. S2: Cells were treated with MLN8237 for different time points followed by SA-β-Gal staining. The percentage of SA-β-Gal positive cells is shown. Fig. S3: Cells were treated with increased concentration of the DR5 small-molecule activator and DMSO or MLN8237 for five days, followed by crystal violet staining. Fig. S4: The tissue slides from the PDX experiment (IgG, DR5 Ab, MLN8237 or MLN8237 + DR5 Ab treatment)were stained for GLB1 (Green) and cleaved caspase 3 (Red).

Supplementary figure legends

Supplementary Figure S6. Results of the flow cytometric analysis of indicated populations of leukocytes in spleen (A) and bone marrow (B) of C57Bl/6 mice bearing B16F0 melanoma tumors.