A new Neu-a syngeneic model of spontaneously metastatic HER2-positive breast cancer.

Metastatic disease results from the dissemination of tumor cells beyond their organ of origin to grow in distant organs and is the primary cause of death in patients with advanced breast cancer. Preclinical murine models in which primary tumors spontaneously metastasize are valuable tools for studying metastatic progression and novel cancer treatment combinations. Here, we characterize a novel syngeneic murine breast tumor cell line that provides a model of spontaneously metastatic neu-expressing breast cancer with quicker onset of widespread metastases after orthotopic mammary implantation in immune-competent NeuN mice. The NT2.5-lung metastasis (-LM) cell line was derived from serial passaging of tumor cells that were macro-dissected from spontaneous lung metastases after orthotopic mammary implantation of parental NT2.5 cells. Within one week of NT2.5-LM implantation, metastases are observed in the lungs. Within four weeks, metastases are also observed in the bones, spleen, colon, and liver. We demonstrate that NT2.5-LM metastases are positive for NeuN-the murine equivalent of human epidermal growth factor 2 (HER2). We further demonstrate altered expression of markers of epithelial-to-mesenchymal transition (EMT), suggestive of their enhanced metastatic potential. Genomic analyses support these findings and reveal enrichment in EMT-regulating pathways. In addition, the metastases are rapidly growing, proliferative, and responsive to HER2-directed therapy. The new NT2.5-LM model provides certain advantages over the parental NT2/NT2.5 model, given its more rapid and spontaneous development of metastases. Besides investigating mechanisms of metastatic progression, this new model may be used for the rationalized development of novel therapeutic interventions and assessment of therapeutic responses.

Mimicking the breast metastatic microenvironment: characterization of a novel syngeneic model of HER2+ breast cancer.

Preclinical murine models in which primary tumors spontaneously metastasize to distant organs are valuable tools to study metastatic progression and novel cancer treatment combinations. Here, we characterize a novel syngeneic murine breast tumor cell line, NT2.5-lung metastasis (-LM), that provides a model of spontaneously metastatic neu-expressing breast cancer with quicker onset of widespread metastases after orthotopic mammary implantation in immune-competent NeuN mice. Within one week of orthotopic implantation of NT2.5-LM in NeuN mice, distant metastases can be observed in the lungs. Within four weeks, metastases are also observed in the bones, spleen, colon, and liver. Metastases are rapidly growing, proliferative, and responsive to HER2-directed therapy. We demonstrate altered expression of markers of epithelial-to-mesenchymal transition (EMT) and enrichment in EMT-regulating pathways, suggestive of their enhanced metastatic potential. The new NT2.5-LM model provides more rapid and spontaneous development of widespread metastases. Besides investigating mechanisms of metastatic progression, this new model may be used for the rationalized development of novel therapeutic interventions and assessment of therapeutic responses targeting distant visceral metastases.

Fibroblasts in the Aged Pancreas Drive Pancreatic Cancer Progression.

Pancreatic cancer is more prevalent in older individuals and often carries a poorer prognosis for them. The relationship between the microenvironment and pancreatic cancer is multifactorial, and age-related changes in nonmalignant cells in the tumor microenvironment may play a key role in promoting cancer aggressiveness. Because fibroblasts have profound impacts on pancreatic cancer progression, we investigated whether age-related changes in pancreatic fibroblasts influence cancer growth and metastasis. Proteomics analysis revealed that aged fibroblasts secrete different factors than young fibroblasts, including increased growth/differentiation factor 15 (GDF-15). Treating young mice with GDF-15 enhanced tumor growth, whereas aged GDF-15 knockout mice showed reduced tumor growth. GDF-15 activated AKT, rendering tumors sensitive to AKT inhibition in an aged but not young microenvironment. These data provide evidence for how aging alters pancreatic fibroblasts and promotes tumor progression, providing potential therapeutic targets and avenues for studying pancreatic cancer while accounting for the effects of aging. SIGNIFICANCE: Aged pancreatic fibroblasts secrete GDF-15 and activate AKT signaling to promote pancreatic cancer growth, highlighting the critical role of aging-mediated changes in the pancreatic cancer microenvironment in driving tumor progression. See related commentary by Isaacson et al., p. 1185.

Mesothelin CAR T Cells Secreting Anti-FAP/Anti-CD3 Molecules Efficiently Target Pancreatic Adenocarcinoma and its Stroma.

PURPOSE: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). EXPERIMENTAL DESIGN: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. RESULTS: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CONCLUSIONS: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.

CD4 T cell-activating neoantigens enhance personalized cancer vaccine efficacy.

Personalized cancer vaccines aim to activate and expand cytotoxic antitumor CD8+ T cells to recognize and kill tumor cells. However, the role of CD4+ T cell activation in the clinical benefit of these vaccines is not well defined. We previously established a personalized neoantigen vaccine (PancVAX) for the pancreatic cancer cell line Panc02, which activates tumor-specific CD8+ T cells but required combinatorial checkpoint modulators to achieve therapeutic efficacy. To determine the effects of neoantigen-specific CD4+ T cell activation, we generated a vaccine (PancVAX2) targeting both major histocompatibility complex class I- (MHCI-) and MHCII-specific neoantigens. Tumor-bearing mice vaccinated with PancVAX2 had significantly improved control of tumor growth and long-term survival benefit without concurrent administration of checkpoint inhibitors. PancVAX2 significantly enhanced priming and recruitment of neoantigen-specific CD8+ T cells into the tumor with lower PD-1 expression after reactivation compared with the CD8+ vaccine alone. Vaccine-induced neoantigen-specific Th1 CD4+ T cells in the tumor were associated with decreased Tregs. Consistent with this, PancVAX2 was associated with more proimmune myeloid-derived suppressor cells and M1-like macrophages in the tumor, demonstrating a less immunosuppressive tumor microenvironment. This study demonstrates the biological importance of prioritizing and including CD4+ T cell-specific neoantigens for personalized cancer vaccine modalities.

The association between the 2D:4D ratio and psychopathic characteristics.

Prior research has identified relations between prenatal testosterone exposure and various antisocial and criminal behaviors. However, less is known about the association between prenatal testosterone exposure and personality traits, such as psychopathy. This study used self-report and biometric data from a sample of undergraduates (n = 491) at a large southwestern university to examine the association between prenatal testosterone exposure (measured by the 2D:4D ratio) and three dimensions of psychopathy (i.e., callousness, egocentricity, and antisocial behavior). Analyses were stratified by sex to explore sex-specific biological underpinnings of psychopathy in young adulthood. Results showed that males scored significantly higher in psychopathic traits and reported significantly lower 2D:4D ratios, compared to females. Additionally, 2D:4D ratios were negatively associated with egocentricity in males, but not females. These findings contribute to a growing literature on the organizational effects that prenatal testosterone exposure may have on the development of different dimensions of psychopathy.

Testosterone, cortisol, and criminal behavior in men and women.

Only two studies to date have considered the joint effects of testosterone and cortisol on direct measures of criminal behavior. The current study extends this earlier work by incorporating the direct and interactive effects of baseline hormone measures and hormone change scores in response to social stress. The current study also extends prior work by considering distinct measures of different criminal behavior types and sex differences. Analyses based on a large sample of undergraduates indicated that testosterone had a positive and statistically significant association with impulsive and violent criminal behavior. The interaction of testosterone with cortisol had a negative association with income generating crime. Simple slopes analyses of this interaction indicated testosterone had a positive association with income generating crime when cortisol was low (-1 SD). Associations between hormones and criminal behavior were not moderated by sex.

Testing the role of inflammation in the relation of childhood maltreatment and suicidal ideation among young adults.

BACKGROUND: In recent years, the prevalence of suicidal ideation among young adults has been on the rise, with childhood maltreatment thought to partially explain this disparity. Systemic inflammation-a product of over-activation of the body's stress response system-has been hypothesized to play a predictive role in the development of suicidal ideation. Enduring childhood maltreatment can lead to systemic inflammation, possibly accounting for suicidal ideation's increased prevalence among young adults who have a history of childhood maltreatment. METHODS: The current study sought to investigate the importance of childhood maltreatment as a static risk factor for downstream suicidal ideation in young adulthood with the immunological response (i.e., systemic inflammation) to childhood maltreatment serving as a mediating factor. RESULTS: Systemic inflammation was found to be positively associated with suicidal ideation, supporting the unique role systemic inflammation may play in the pathogenesis of suicidal ideation, though hypotheses regarding childhood maltreatment were not supported. CONCLUSION: This study provides novel insight into a potential immunobiological model for suicidal ideation development in young adult populations.

Examining the Relationship Between Victimization, Psychopathy, and the Acceptance of Rape Myths.

Rape myths are attitudes that implicitly and explicitly blame victims for their own sexual victimization. Greater adherence to rape myths is linked to several negative outcomes, including the neutralization of gender-based violence and the perpetration of sexual violence. Few studies have considered how previous life experiences and individual-level traits influence the development and greater adherence to rape myths. The current study examines how traits associated with the three-factor model of psychopathy (i.e., egocentric, callous, and antisocial dimensions) and adherence to traditional gender roles mediate the relationship between prior childhood/adolescent victimization and the acceptance of rape myths in a sample of college men and women (N = 789). Path modeling indicates that experiences of psychological victimization (before age 16) increased egocentric psychopathic traits, which then increased the acceptance of rape myths in men. In women, however, sexual victimization (before age 16) increased the acceptance of traditional gender roles, which then influenced the acceptance of rape myths. Additionally, the egocentric facet of psychopathy exerted indirect effects on the acceptance of rape myths through traditional views on gender roles in both men and women. These findings highlight the need to continue to examine egocentric personality traits in relation to the development of rape myths in adolescent and young adult populations. Directions for collegiate programming are discussed.

Inflammatory and environmental contributions to social information processing.

It appears that social information processing is negatively affected by inflammation, but extant research is primarily experimental and comes from laboratory-based manipulations of inflammatory states. We aimed to examine interactions between inflammation, stressful life events, and positive memories of childhood relations with parents in relation to social information processing in 201 adults. We hypothesized that increased inflammation and stressful life events would be associated with greater hostile social information processing, but that positive memories of childhood relations with parents would moderate both relations. Results indicated that high IL-6 levels and stressful life events were significantly associated with direct and hostile social information processing. Positive memories of childhood relations with parents attenuated the link between stressful life events and social information processing. Findings suggest that both immune function and environmental stressors are related to social information processing and that positive memories of childhood relations exert some buffering effect.

Implantation of a neoantigen-targeted hydrogel vaccine prevents recurrence of pancreatic adenocarcinoma after incomplete resection.

Tumor involvement of major vascular structures limits surgical options in pancreatic adenocarcinoma (PDAC), which in turn limits opportunities for cure. Despite advances in locoregional approaches, there is currently no role for incomplete resection. This study evaluated a gelatinized neoantigen-targeted vaccine applied to a grossly positive resection margin in preventing local recurrence. Incomplete surgical resection was performed in mice bearing syngeneic flank Panc02 tumors, leaving a 1 mm rim adherent to the muscle bed. A previously validated vaccine consisting of neoantigen peptides, a stimulator of interferon genes (STING) agonist and AddaVaxTM (termed PancVax) was embedded in a hyaluronic acid hydrogel and applied to the tumor bed. Tumor remnants, regional lymph nodes, and spleens were analyzed using histology, flow cytometry, gene expression profiling, and ELISPOT assays. The immune microenvironment at the tumor margin after surgery alone was characterized by a transient influx of myeloid-derived suppressor cells (MDSCs), prolonged neutrophil influx, and near complete loss of cytotoxic T cells. Application of PancVax gel was associated with enhanced T cell activation in the draining lymph node and expansion of neoantigen-specific T cells in the spleen. Mice implanted with PancVax gel demonstrated no evidence of residual tumor at two weeks postoperatively and healed incisions at two months postoperatively without local recurrence. In summary, application of PancVax gel at a grossly positive tumor margin led to systemic expansion of neoantigen-specific T cells and effectively prevented local recurrence. These findings support further work into locoregional adjuncts to immune modulation in PDAC.

Systemic inhibition of PTPN22 augments anticancer immunity.

Both epidemiologic and cellular studies in the context of autoimmune diseases have established that protein tyrosine phosphatase non-receptor type 22 (PTPN22) is a key regulator of T cell receptor (TCR) signaling. However, its mechanism of action in tumors and its translatability as a target for cancer immunotherapy have not been established. Here we show that a germline variant of PTPN22, rs2476601, portended a lower likelihood of cancer in patients. PTPN22 expression was also associated with markers of immune regulation in multiple cancer types. In mice, lack of PTPN22 augmented antitumor activity with greater infiltration and activation of macrophages, natural killer (NK) cells, and T cells. Notably, we generated a novel small molecule inhibitor of PTPN22, named L-1, that phenocopied the antitumor effects seen in genotypic PTPN22 knockout. PTPN22 inhibition promoted activation of CD8+ T cells and macrophage subpopulations toward MHC-II expressing M1-like phenotypes, both of which were necessary for successful antitumor efficacy. Increased PD1-PDL1 axis in the setting of PTPN22 inhibition could be further leveraged with PD1 inhibition to augment antitumor effects. Similarly, cancer patients with the rs2476601 variant responded significantly better to checkpoint inhibitor immunotherapy. Our findings suggest that PTPN22 is a druggable systemic target for cancer immunotherapy.

Multi-omic profiling of lung and liver tumor microenvironments of metastatic pancreatic cancer reveals site-specific immune regulatory pathways.

BACKGROUND: The majority of pancreatic ductal adenocarcinomas (PDAC) are diagnosed at the metastatic stage, and standard therapies have limited activity with a dismal 5-year survival rate of only 8%. The liver and lung are the most common sites of PDAC metastasis, and each have been differentially associated with prognoses and responses to systemic therapies. A deeper understanding of the molecular and cellular landscape within the tumor microenvironment (TME) metastasis at these different sites is critical to informing future therapeutic strategies against metastatic PDAC. RESULTS: By leveraging combined mass cytometry, immunohistochemistry, and RNA sequencing, we identify key regulatory pathways that distinguish the liver and lung TMEs in a preclinical mouse model of metastatic PDAC. We demonstrate that the lung TME generally exhibits higher levels of immune infiltration, immune activation, and pro-immune signaling pathways, whereas multiple immune-suppressive pathways are emphasized in the liver TME. We then perform further validation of these preclinical findings in paired human lung and liver metastatic samples using immunohistochemistry from PDAC rapid autopsy specimens. Finally, in silico validation with transfer learning between our mouse model and TCGA datasets further demonstrates that many of the site-associated features are detectable even in the context of different primary tumors. CONCLUSIONS: Determining the distinctive immune-suppressive features in multiple liver and lung TME datasets provides further insight into the tissue specificity of molecular and cellular pathways, suggesting a potential mechanism underlying the discordant clinical responses that are often observed in metastatic diseases.

An exploratory analysis of testosterone, cortisol, and aggressive behavior type in men and women.

Increasing evidence indicates that the interaction between testosterone and cortisol is associated with variation in aggressive behavior. However, results are mixed. The current study further explored the association between testosterone, cortisol, and both reactive and proactive aggression in a large sample of university students. Models considered direct and interactive effects between baseline measures of testosterone and cortisol as well as change in hormones in response to a social stressor. In women, baseline cortisol had a negative direct association with reactive aggression and was further associated with reactive aggression in interaction with baseline testosterone (positive interaction). Hormones were unrelated to reactive aggression in men. Baseline cortisol had a negative direct association with proactive aggression in women. In contrast, the association between change in cortisol and proactive aggression was positive. Cortisol was not associated with proactive aggression in men. In addition, testosterone was not related to proactive aggression either directly or in interaction with cortisol in either men or women. Collectively, these results show that the association between hormones and aggression varies across aggressive behavior type and across sex.

Further Evaluation of the Associations Between Psychopathic Traits and Symptoms of PTSD and Depression in a Nonclinical Sample.

Examining psychopathic traits at the factor or facet level has revealed that various aspects of psychopathy may be differentially related, even in opposing directions, to important outcomes (e.g., intelligence, emotion regulation). Empirical work on relations between psychopathy and internalizing disorders, such as posttraumatic stress disorder (PTSD) and depression, has provided evidence for a positive association with antisocial traits. However, findings for the affective domain have been more equivocal. The current study (N = 732) sought to replicate past findings of the positive association of antisocial psychopathic traits with higher levels of PTSD and depressive symptoms, and to further explore associations between affective traits of psychopathy and these disorders using two measures of psychopathy. Results confirmed prior findings of a positive correlation between antisocial features and self-reported PTSD/Depression symptom severity, but they did not provide evidence for any association with affective traits. Future research using longitudinal designs is needed to begin establishing temporal ordering of the psychopathy-internalizing relationship.

Role of in silico structural modeling in predicting immunogenic neoepitopes for cancer vaccine development.

In prior studies, we delineated the landscape of neoantigens arising from nonsynonymous point mutations in a murine pancreatic cancer model, Panc02. We developed a peptide vaccine by targeting neoantigens predicted using a pipeline that incorporates the MHC binding algorithm NetMHC. The vaccine, when combined with immune checkpoint modulators, elicited a robust neoepitope-specific antitumor immune response and led to tumor clearance. However, only a small fraction of the predicted neoepitopes induced T cell immunity, similarly to that reported for neoantigen vaccines tested in clinical studies. While these studies have used binding affinities to MHC I as surrogates for T cell immunity, this approach does not include spatial information on the mutated residue that is crucial for TCR activation. Here, we investigate conformational alterations in and around the MHC binding groove induced by selected minimal neoepitopes, and we examine the influence of a given mutated residue as a function of its spatial position. We found that structural parameters, including the solvent-accessible surface area (SASA) of the neoepitope and the position and spatial configuration of the mutated residue within the sequence, can be used to improve the prediction of immunogenic neoepitopes for inclusion in cancer vaccines.

Inhibition of miR-21 Regulates Mutant KRAS Effector Pathways and Intercepts Pancreatic Ductal Adenocarcinoma Development.

Almost all pancreatic ductal adenocarcinomas (PDA) develop following KRAS activation, which triggers epithelial transformation and recruitment of desmoplastic stroma through additional transcriptional and epigenetic regulation, but only a few of these regulatory mechanisms have been described. We profiled dysregulated miRNAs starting with the earliest premalignant pancreatic intraepithelial neoplasias (PanIN) in genetically engineered mutated KRAS and P53 (KPC) mice programmed to recapitulate human PDA tumorigenesis. We identified miR-21 and miR-224 as cell-specific and compartment-specific regulators in PanINs and PDA. miR-21 is overexpressed in tumor epithelial cells of premalignant ducts, while miR-224 is overexpressed in cancer-associated fibroblasts in PDA stroma. Inhibition of miR-21 reverted protumorigenic functionalities to baseline levels. Overexpression of miR-224 induced activated phenotypes in normal fibroblasts. In vivo miR-21 inhibition improved survival in established PDA. Importantly, early systemic miR-21 inhibition completely intercepted premalignant progression. Finally, an evaluation of miR-21 expression in the PDA cohort of The Cancer Genome Atlas identified a correlation between tumor epithelial cell content and miR-21 expression in human tumors providing further rationale for conducting human studies. Thus, miR-21 may be useful for early PanIN detection, and for intercepting developing premalignant pancreatic lesions and other KRAS-driven premalignancies.

Effects of B cell-activating factor on tumor immunity.

Immunotherapies that modulate T cell function have been firmly established as a pillar of cancer therapy, whereas the potential for B cells in the antitumor immune response is less established. B cell-activating factor (BAFF) is a B cell-activating cytokine belonging to the TNF ligand family that has been associated with autoimmunity, but little is known about its effects on cancer immunity. We find that BAFF upregulates multiple B cell costimulatory molecules; augments IL-12a expression, consistent with Be-1 lineage commitment; and enhances B cell antigen-presentation to CD4+ Th cells in vitro. In a syngeneic mouse model of melanoma, BAFF upregulates B cell CD40 and PD-L1 expression; it also modulates T cell function through increased T cell activation and TH1 polarization, enhanced expression of the proinflammatory leukocyte trafficking chemokine CCR6, and promotion of a memory phenotype, leading to enhanced antitumor immunity. Similarly, adjuvant BAFF promotes a memory phenotype of T cells in vaccine-draining lymph nodes and augments the antitumor efficacy of whole cell vaccines. BAFF also has distinct immunoregulatory functions, promoting the expansion of CD4+Foxp3+ Tregs in the spleen and tumor microenvironment (TME). Human melanoma data from The Cancer Genome Atlas (TCGA) demonstrate that BAFF expression is positively associated with overall survival and a TH1/IFN-γ gene signature. These data support a potential role for BAFF signaling as a cancer immunotherapy.

Nanoparticle interactions with immune cells dominate tumor retention and induce T cell-mediated tumor suppression in models of breast cancer.

The factors that influence nanoparticle fate in vivo following systemic delivery remain an area of intense interest. Of particular interest is whether labeling with a cancer-specific antibody ligand ("active targeting") is superior to its unlabeled counterpart ("passive targeting"). Using models of breast cancer in three immune variants of mice, we demonstrate that intratumor retention of antibody-labeled nanoparticles was determined by tumor-associated dendritic cells, neutrophils, monocytes, and macrophages and not by antibody-antigen interactions. Systemic exposure to either nanoparticle type induced an immune response leading to CD8+ T cell infiltration and tumor growth delay that was independent of antibody therapeutic activity. These results suggest that antitumor immune responses can be induced by systemic exposure to nanoparticles without requiring a therapeutic payload. We conclude that immune status of the host and microenvironment of solid tumors are critical variables for studies in cancer nanomedicine and that nanoparticle technology may harbor potential for cancer immunotherapy.

Robust antigen-specific CD8 T cell tolerance to a model prostate cancer neoantigen.

Immunotherapy has shown limited success in prostate cancer; this may be partially explained by its immunosuppressive tumor microenvironment (TME). Although androgen-deprivation therapy (ADT), the most common treatment for prostate cancer, initially promotes a robust T cell infiltrate, T cell responses are later attenuated. Based on the castration-sensitive Myc-CaP model, we developed an antigen-specific system to study CD8 T cell tolerance to prostate tumors. This model is unique in that CD8 T cells recognize a bona-fide tumor antigen (Her-2/neu), rather than an overexpressed xenogenic antigen like chicken ovalbumin or influenza hemagglutinin. Using this novel model, we demonstrate robust tolerance that is not alleviated by TLR agonists or ADT. This model may serve as a novel and useful tool to further interrogate methods by which to augment anti-tumor cancer immune responses to prostate cancer. Significance: Prostate cancer is a leading cause of cancer-related death in men worldwide, with an estimated 33,000 deaths projected in the U.S. in 2020. Although primary (localized) tumors can be cured by surgery or radiation, approximately 40% of patients eventually develop recurrent disease. While initially responsive to androgen-deprivation, many patients with recurrent prostate cancer eventually progress to a more advanced disease state known as metastatic castration-resistant prostate cancer (mCRPC); this is the lethal phenotype. These studies describe a novel androgen-responsive murine cell line that expresses a bona-fide tumor antigen (Her-2/neu). Pre-clinical work with this model shows robust and antigen-specific CD8 T cell tolerance, providing a novel preclinical model to study CD8 T cell tolerance to prostate tumors.