Aggressive Tumor Growth Research Articles

Loss of testosterone leads to impaired T cell function via glucocorticoids in mouse glioblastoma models

Abstract While sex differences in glioblastoma (GBM) are well established, the contribution of sex hormones on these differences has not been fully explored. Recently, the immunoregulatory effect of testosterone in other type of cancers has been reported. To evaluate the effect of testosterone on GBM, we performed castration or sham surgery on mice, followed by intracranial implantation of syngeneic glioma cells. Surprisingly, the castrated B6 mice had more aggressive tumors compared to sham surgery controls and the castration induced aggressive tumor growth could be rescued by exogenous testosterone administration. This was further confirmed by chemical blockade of androgen receptor signaling using enzalutamide. In contrast, castration in Rag1KO mouse models did not show a survival difference compared to controls, suggesting a role for lymphocytes. Interestingly, upregulated levels of circulating corticosterone in castrated mice was detected by mass spectrometry, and glucocorticoid receptor blocker reversed the survival of castrated mice, suggesting glucocorticoid-mediated systemic immunosuppression upon castration. Flow analysis revealed decreased cytokine production in CD4+ and CD8+ T cells from tumors and lymph nodes in castrated mice. We also found that tumor growth was delayed in castrated mice when tumor cells were subcutaneously implanted in the flank while decreased cytokine production in T cells in tumor and lymph nodes from flank tumor model as well. These data suggest brain-specific effect of steroid hormone on tumor control. Taken together, our findings demonstrate that testosterone regulates T cell function via glucocorticoids, and ultimately affects GBM tumor growth in a site-specific manner. Supported by grants from NIH (R35 NS127083, P01 CA245705).

Resection of Skin Cancer Resulting in Free Vascularized Tissue Reconstruction: Always a Therapeutic Failure?

The globally increasing incidence of cutaneous malignancies leads, in parallel, to increasing numbers of locally advanced skin cancer resulting in reconstructive surgery. Reasons for locally advanced skin cancer may be a patient's neglect or aggressive tumor growth, such as desmoplastic growth or perineural invasion. This study investigates characteristics of cutaneous malignancies requiring microsurgical reconstruction with the aim of identifying possible pitfalls and improving diagnostic and therapeutic processes. A retrospective data analysis from 2015 to 2020 was conducted. Seventeen patients (n = 17) were included. The mean age at reconstructive surgery was 68.5 (±13) years. The majority of patients (14/17, 82%) presented with recurrent skin cancer. The most common histological entity was squamous cell carcinoma (10/17, 59%). All neoplasms showed at least one of the following histopathological characteristics: desmoplastic growth (12/17, 71%), perineural invasion (6/17, 35%), or tumor thickness of at least 6 mm (9/17, 53%). The mean number of surgical resections until cancer-free resection margins (R0) were achieved was 2.4 (±0.7). The local recurrence rate and the rate of distant metastases were 36%. Identified high-risk neoplastic characteristics, such as desmoplastic growth, perineural invasion, and a tumor depth of at least 6 mm, require a more extensive surgical treatment without concerns about defect size.

NOTCH pathway inactivation reprograms stem-like oral cancer cells to JAK-STAT dependent state and provides the opportunity of synthetic lethality

BackgroundWe have recently provided the evidence of interconvertible cellular states, driving non-genetic heterogeneity among stem-like oral cancer cells (oral-SLCCs). Here, NOTCH pathway-activity status is explored as one of the possible mechanisms behind this stochastic plasticity. MethodsOral-SLCCs were enriched in 3D-spheroids. Constitutively-active and inactive status of NOTCH pathway was achieved by genetic or pharmacological approaches. RNA sequencing and real-time PCR was performed for gene expression studies. in vitro cytotoxicity assessments were performed by AlamarBlue assay and in vivo effects were studied by xenograft growth in zebrafish embryo. ResultsWe have observed stochastic plasticity in oral-SLCCs, spontaneously maintaining both NOTCH-active and inactive states. While cisplatin refraction was associated with post-treatment adaptation to the active-state of NOTCH pathway, oral-SLCCs with inactive NOTCH pathway status showed aggressive tumor growth and poor prognosis. RNAseq analysis clearly suggested the upregulation of JAK-STAT pathway in NOTCH pathway-inactive subset. The 3D-spheroids with lower NOTCH-activity status displayed significantly higher sensitivity to JAK-selective drugs, Ruxolitinib or Tofacitinib or siRNA mediated downregulation of tested partners STAT3/4. Oral-SLCCs were programmed to adapt the inactive status of NOTCH pathway by exposing to γ-secretase inhibitors, LY411575 or RO4929097, followed by targeting with JAK-inhibitors, Ruxolitinib or Tofacitinib. This approach resulted in a very significant inhibition in viability of 3D-spheroids as well as xenograft initiation in Zebrafish embryos. ConclusionStudy revealed for the first time that NOTCH pathway-inactive state exhibit activation of JAK-STAT pathways, as synthetic lethal pair. Therefore, co-inhibition of these pathway may serve as novel therapeutic strategy against aggressive oral cancer.

Abstract 272: Autophagy and MEK inhibition promotes ferroptosis in liver kinase B1 (Lkb1)-deficient Kras-driven lung tumors

Abstract Tumor suppressor Liver Kinase B1 (LKB1) activates 5’-adenosine monophosphate protein kinase (AMPK) and maintains energy homeostasis in response to energy crises. LKB1 and KRAS are the third most frequent co-mutations detected in non-small cell lung cancer (NSCLC), causing aggressive tumor growth and metastases. Unfortunately, standard treatment with RAS-RAF-MEK-ERK signaling pathway inhibitors has minimal therapeutic efficacy in LKB1-mutant KRAS-driven NSCLC. Thus, identifying a novel treatment for patients harboring co-mutations in LKB1 and KRAS is urgently needed. Autophagy degrades and recycles the building blocks for cancer cells to survive metabolic challenges. Using genetically engineered mouse models (GEMMs), we have previously demonstrated that autophagy compensates for Lkb1 loss for Kras-driven lung tumorigenesis; loss of an autophagy-essential gene Atg7 dramatically impaired tumor initiation and tumor growth in KrasG12D/+;Lkb1−/− (KL) lung tumors. This is in sharp contrast to Lkb1 wild-type (WT) (KrasG12D/+;p53−/− (KP)) tumors that are less sensitive to autophagy gene ablation. To further value our discoveries in clinical translational ability, we treated mouse lung tumor derived cell lines (TDCLs) with FDA-approved autophagy inhibitor hydroxychloroquine (HCQ) and MEK inhibitor Trametinib and found that the combination treatment displayed synergistic anti-proliferative effects in KL TDCLs compared to KP TDCLs. To elucidate the underlying mechanism of increased sensitivity of KL TDCLs to Trametinib by autophagy ablation, we performed metabolomic profiling of KL TDCLs with Trametinib, HCQ, or combination treatment and found that several glycolytic and TCA cycle intermediates, amino acids, and ATP levels were significantly upregulated upon treatment with Trametinib, which were significantly reduced by the combination treatment. In addition, the combination treatment significantly reduced mitochondrial membrane potential, basal respiration, and ATP production in KL TDCLs. In vivo studies using tumor allografts, genetically engineered mouse models (GEMMs) and patient-derived xenografts (PDXs) showed anti-tumor activity of the combination treatment on KL tumors, but not in KP tumors. Moreover, we found increased lipid peroxidation indicative of ferroptosis in KL TDCLs and KL PDX tumors with the combination treatment compared to the single agent treatments. Finally, treatment with a ferroptosis inhibitor rescued the reduced KL allograft tumor growth caused by the combination treatment. Taken together, our observations indicate that autophagy upregulation in KL tumors causes resistance to Trametinib treatment by maintaining energy homeostasis for cell survival and inhibits ferroptosis. Therefore, a combination of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat LKB1-deficient KRAS-driven NSCLC. Citation Format: Vrushank Bhatt, Taijin Lan, Wenping Wang, Jerry Kong, Eduardo Cararo Lopes, Khoosheh Khayati, Jianming Wang, Akash Raju, Michael Rangel, Enrique Lopez, Zhixian Sherrie Hu, Xuefei Luo, Xiaoyang Su, Jyoti Malhotra, Wenwei Hu, Sharon R. Pine, Eileen White, Jessie Yanxiang Guo. Autophagy and MEK inhibition promotes ferroptosis in liver kinase B1 (Lkb1)-deficient Kras-driven lung tumors [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 272.

Abstract 2256: Cancer stem cells and exosomal PD-L1 suppress anti-tumor B cells

Abstract We have conducted clinical trials for cancer immunotherapy using vaccine-primed lymph node (VPLN) T calls based on our animal studies with tumor-draining lymph node (TDLN) T cells. More recently we have identified antitumor effector TDLN B cells which demonstrated significant tumor reactivity in vitro and therapeutic efficacy in vivo upon adoptive transfer which can be enhanced by anti-PD-L1 administration. It is unknown whether tumor cells, particularly cancer stem cells (CSCs) could suppress the antitumor effector B cells. We found that depletion of B cells with anti-CD20 mAb prior to anti-PD-L1 administration resulted in more aggressive tumor growth, and the anti-tumor efficacy of anti-PD-L1 was significantly reduced in both 4T1 and CT26 murine tumor-bearing hosts, suggesting the involvement of host B cells in the anti-tumor effect of anti-PD-L1. In addition, administration of anti-PD-L1 partially recovered the humoral immune response, confirming the PD-L1/PD-1 pathway involvement in B cell suppression. We detected elevated expression of PD-1 on activated B cells, and higher PD-L1 expression on ALDHhigh CSCs than on ALDHlow non-CSCs. Co-culturing ALDHhigh CSCs with purified B cells significantly reduced IgG secretion, and such CSC-mediated B cell suppression was rescued by adding anti-PD-L1 mAb in a dose-dependent manner to the CSC-B cell co-culture. These experiments thus indicate that CSCs suppress the IgG production by B cells via the PD-L1/PD-1 axis. Tumor cells were recently recognized to suppress T cell function via secretion of exosomes that express PD-L1. To investigate if exosomal PD-L1 contributes to B cell immunosuppression, we generated Rab27ako D5 cells to delete exosomes as well as PD-L1ko D5 cells by CRISPR/Cas9 gene editing. D5-derived exosomes suppressed B cell proliferation and IgG production in vitro, and administration of these exosomes promoted tumor growth and reduced animal survival, revealing the role of tumor-derived exosomes in B cell immunosuppression. Tumor-derived exosomes expressed PD-L1, but PD-L1 was absent in the exosomes isolated from the PD-L1ko tumor cells as evident by western blot and flow cytometry. Deletion of Rab27a or PD-L1 did not affect the proliferation of the tumor cells in vitro. However, Rab27ako or PD-L1ko resulted in significantly enhanced host anti-tumor immunity evident by reduced 4T1 tumor growth in vivo compared with the WT tumor. Importantly, anti-PD-L1 therapy significantly reduced Rab27ako or PD-L1ko 4T1 tumor growth and prolonged animal survival vs. the WT tumor control. We conclude that PD-L1-expresing CSCs and PD-L1-expresing exosomes suppress antitumor effector B cells via the PD-L1/PD-1 axis. To characterize the suppression of CSC-derived exosomes on host B cells as well as T cells, isolation of Rab27ako ALDHhigh CSCs warranties further investigation. Citation Format: Ying Xin, Jiayu Liu, You Qin, Kexing Lyu, Sifei Yu, Ming Lin, Max Wicha, Alfred E. Chang, Qiao Li. Cancer stem cells and exosomal PD-L1 suppress anti-tumor B cells [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 2256.

Abstract 1729: MG53 suppresses the tumor growth via transcriptional inhibiting the expression of KIF11 in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with limited therapeutic options and a dismal long-term survival. The upregulation of KIF11, a motor protein that regulates cell mitosis, has been associated with the aggressive proliferation of pancreatic cancer cells. A molecular strategy to counteract the hyperactivity of KIF11 may represent a potential means to treat PDAC. We previously identified MG53, a member of the tripartite motif family protein (TRIM72), as an important component of tissue repair, and revealed its potential application in regenerative medicine. So far, most published studies have focused on the cytosolic and vesicle-trafficking function of MG53, little is known on the nuclear action of MG53 in pancreatic tumor suppression. In the present study, our confocal imaging revealed frequent appearance of MG53 in the nucleus of human pancreatic cancer cells, and subcellular fractionation assay showed that a majority of MG53 protein was present in the nuclear fraction. Decreased MG53 and increased KIF11 protein expression were observed in both pancreatic cancer cell lines (PANC-1 and mPanc96) and PDAC tissue from surgically resected tumors. Overexpressing MG53 inhibited colony formation and proliferation of PANC-1 cells with significant reduction of KIF11 expression. Furthermore, we demonstrated that MG53-mediated control of KIF11 expression occurs at the gene transcription level. Chromatin immunoprecipitation demonstrated an interaction between MG53 and the promoter of KIF11 in PANC-1 cells. Using a luciferase cell reporter driven by the KIF11 promoter, we found that overexpressing MG53 led to downregulation of the KIF11 transcription activity, and induced cell cycle arrest at the G2/M phase. We also conducted syngeneic orthotopic transplantation of a mouse pancreatic cancer cell line, KPC cells derived from pancreatic tumor in KRasG12D/Trp53-/-/Pdx-1-Cre (KPC) mice, into the pancreas of wild type (WT) and mg53-/- (MG53-KO) mice, which showed aggressive pancreatic tumor growth in MG53-KO mice compared with WT mice after 3-week of transplantation. Western blots revealed that a considerable amount of MG53 protein was found in the pancreatic tumors derived from KPCs in WT mice vs. those in MG53-KO mice, indicating the accumulation of circulating MG53 during tumor growth. Overall, these findings demonstrated that MG53 functions as a tumor suppressor to inhibit cancer cell proliferation and the tumor growth through transcriptional down-regulating the expression of KIF11 in pancreatic cancer, and provided a new therapeutic target for the treatment of patients with PDAC. Citation Format: Xiao-Liang Wang, Xiangfei He, Zobeida Cruz-Monserrate, Allen Tsung, Jianjie Ma, Chuanxi Cai. MG53 suppresses the tumor growth via transcriptional inhibiting the expression of KIF11 in pancreatic 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 1729.

Association Between the Immunohistochemistry Expression of E-cadherin, Beta-Catenin, and CD44 in Colorectal Adenocarcinoma.

Background Colorectal cancer is a leading cause of cancer-related deaths worldwide, and epithelial-mesenchymal transition (EMT) plays an important role in cancer metastasis. In EMT, there is downregulation of E-cadherin, an intracellular adhesion molecule, as well as mutations in beta-catenin genes. On immunohistochemistry (IHC), the expression of CD44 portrays stem cell differentiation, which, in turn, is strongly associated with EMT. Thus, newer targeted therapies can be advised based on the expression of EMT and stem cell differentiation. Aims and objectives To determine the IHC expression of E-cadherin, beta-catenin, and CD44 in colorectal adenocarcinoma and find the association of the IHC expression of E-cadherin, beta-catenin, and CD44 with the histopathological grade, stage, lymph node metastasis, and lymphovascular invasion of colorectal adenocarcinoma. Materials and methods Fifty histologically proven cases of colorectal adenocarcinoma from 2016 to 2021 were included in this study, and clinicopathological dataincluding age, gender, grading, TNM (tumour, node, and metastasis) staging, and lymph node metastasiswere collected and hematoxylin and eosin slides were reviewed. IHC staining for E-cadherin, beta-catenin, and CD44 was done for all cases using the peroxidase and anti-peroxidase method, and the results were analysed. Results Peak incidence occurred in the 61-70 years age group (36%), and the most common site of the tumour was the rectal area (48%). The majority of the cases were in TNM stage II (37.3%), and a low expression of E-cadherin was found to be associated with higher T stage (p = 0.03), TNM staging (p = 0.04), as well as the presence of lymph node metastasis (p = 0.006). High beta-catenin expression was observed to have a significant correlation with a higher T stage (p = 0.006) and TNM staging (p = 0.005), while high CD44 expression was found to be associated with lymph node metastasis (p = 0.01). Altered expression of EMT-related proteins (E-cadherin and beta-catenin) showed a significant correlation with higher T stage (p = 0.03), TNM staging (p = 0.016), and lymph node metastasis (0.04). Conclusions EMT and cancer stem cell IHC markers are biomarkers for aggressive tumour growth and lymph node metastasis.Hence, EMT markers (E-cadherin and beta-catenin) and cancer stem cell markers (CD44) can be used as prognostic markers.

Tumor Associated Neutrophil Polarization and its Potential Effects in Cancer Therapy

Neutrophils are the first line of defense against pathogens in the immune system, and also has important implications for the tumor micro-environment. The effect of neutrophils on cancer is a double-edged sword in that they can both promote or inhibit tumor growth. When in different tumor environments or exposed to different stimulation, neutrophils would polarize toward anti-tumorigenic N1 or pro-tumorigenic N2 phenotype. The exact mechanism to polarize N1 and N2 are not yet well understood, but it is currently known that IFN-β or stimulation of the pathogen-associated molecular patterns (PAMPs) pathway can cause N1 polarization. On the contrary, in the presence of TGF-β or IL-10, N2 polarization occurs. When N1 population is dominant in the tumor micro-environment, they will inhibit tumor growth and enhance the effect of immunotherapy. But vice versa, dominance of pro-tumorigenic N2 phenotype within the tumor would enhance inflammation leading to more aggressive tumor growth and also hinders the effect of immunotherapy. Therefore, it is very important to understand the subtype of tumor associated neutrophils in the tumor when determining appropriate cancer therapy.

Indices of insulin-like growth factors family in the lung tissue of patients with non-small cell lung cancer after COVID-19 of various severity

Purpose of the study. An analysis of levels of IGF and their carrying proteins in lung tissues of cancer patients depending on the severity of the previous COVID-19 infection.Patients and methods. The study included 60 patients with histologically verified non-small cell lung cancer (NSCLC) T1–3NхM0 receiving treatment at the Thoracic Department, National Medical Research Centre for Oncology, in 2020–2021. The control group included 30 NSCLC patients after asymptomatic or mild COVID-19 disease (15 males and 15 females); the main group included 30 (15 men and 15 women) patients after severe or moderate to severe COVID-19 infection. The mean age of patients was 59.11 ± 2.89 years; no significant differences were noted between the control and main groups. All participants gave their informed consent prior to the study approved by the Ethics Committee of National Medical Research Centre for Oncology. Qualitative assessment of IGF-I, IGF-II and IGFBP-1,2,3 levels in the tissues of the tumor, peritumoral area and resection line were measured by ELISA (Mediagnost, Germany). The statistical analysis was performed in the Statistica 10 program, the differences were considered statistically significant at p < 0.05.Results. Regardless of the gender, levels of IGF-I and IGF-II in tumor and resection line samples in patients of the main group were higher than in the control group on average by 1.5–2.2 times, and IGFBP-1 in the tumor was lower by 1.3 times in men and by 5 times in women. The ratio of IGF and IGFBP-1-3 in patients of the control group in perifocal tissues changed towards the parameters in the tumor tissue. IGF/IGFBP-1-3 in men of the main group were lower or did not differ from the indices in the intact tissue, while in women they increased, similarly to the tumor tissue.Conclusion. An increase in the ratio of IGF and carrier proteins in the tumor tissue of patients in the main group indicated an excessive accumulation of IGF in it, which may contribute to more aggressive growth of malignant tumors. The most pronounced disorders in the system of insulin-like growth factors were found in the tissues of the tumor and intact lung of patients with previous severe and moderate to severe COVID-19.

Inhibition of autophagy and MEK promotes ferroptosis in Lkb1-deficient Kras-driven lung tumors

LKB1 and KRAS are the third most frequent co-mutations detected in non-small cell lung cancer (NSCLC) and cause aggressive tumor growth. Unfortunately, treatment with RAS-RAF-MEK-ERK pathway inhibitors has minimal therapeutic efficacy in LKB1-mutant KRAS-driven NSCLC. Autophagy, an intracellular nutrient scavenging pathway, compensates for Lkb1 loss to support Kras-driven lung tumor growth. Here we preclinically evaluate the possibility of autophagy inhibition together with MEK inhibition as a treatment for Kras-driven lung tumors. We found that the combination of the autophagy inhibitor hydroxychloroquine (HCQ) and the MEK inhibitor Trametinib displays synergistic anti-proliferative activity in KrasG12D/+;Lkb1-/- (KL) lung cancer cells, but not in KrasG12D/+;p53-/- (KP) lung cancer cells. In vivo studies using tumor allografts, genetically engineered mouse models (GEMMs) and patient-derived xenografts (PDXs) showed anti-tumor activity of the combination of HCQ and Trametinib on KL but not KP tumors. We further found that the combination treatment significantly reduced mitochondrial membrane potential, basal respiration, and ATP production, while also increasing lipid peroxidation, indicative of ferroptosis, in KL tumor-derived cell lines (TDCLs) and KL tumors compared to treatment with single agents. Moreover, the reduced tumor growth by the combination treatment was rescued by ferroptosis inhibitor. Taken together, we demonstrate that autophagy upregulation in KL tumors causes resistance to Trametinib by inhibiting ferroptosis. Therefore, a combination of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat NSCLC bearing co-mutations of LKB1 and KRAS.

Connexin 43 trafficking and regulation of gap junctional intercellular communication alters ovarian cancer cell migration and tumorigenesis

Ovarian cancer persists to be the most lethal gynecological malignancy, demanding rigorous treatments involving radio-chemotherapy that trigger toxicity and consequently mortality among patients. An improved understanding of the disease progression may pioneer curative therapies. Mouse epithelial ovarian cancer cell lines, ID8 and ID8-VEGF (overexpressing VEGF) were intraperitoneally injected in C57BL/6 female mice to develop a Syngeneic Ovarian cancer mouse model. It was observed that ID8-VEGF cells were able to induce aggressive tumor growth in mice compared to ID8 cells. Furthermore, results of the current in vitro study comparing ID8 and ID8-VEGF demonstrated that highly tumorigenic ID8-VEGF had reduced gap junctional intercellular communication (GJIC) due to intracellular Connexin 43 (Cx43) expression. Additionally, ID8 cells with reduced tumorigenic capability expressed significant GJIC. Furthermore, loss of GJIC in ID8-VEGF cells induced shorter tunneling nanotube formations, while ID8 cells develops longer tunneling nanotube to maintain cellular crosstalk. The administration of a pharmacological drug 4-phenylbutyrate (4PBA) ensured the restoration of GJIC in both the ovarian cancer cell lines. Additionally, 4PBA treatment significantly inhibited the migration of ovarian cancer cell lines and tumor formation in ovarian cancer mice models. In summary, the 4PBA-mediated restoration of GJIC suppressed migration (in vitro) and tumorigenesis (in vivo) of ovarian cancer cells. The present study suggests that Cx43 assembled GJIC and its supportive signaling pathways are a prospective target for restricting ovarian cancer progression.

Ethnic disparities in the immune microenvironment of triple negative breast cancer and its role in therapeutic outcomes.

In 2020, newly diagnosed breast cancer (BC) cases surpassed that of lung cancer among women, making it the most common female cancer globally. In spite of recent increases in incidence rates, mortality due to BC has declined since 1989. These declines have been attributed to advancements in treatment modalities as well as increased mammography surveillance. Despite these advances, African American (AA) women are 40% more likely to die from BC than Caucasian women. Multifactorial etiology has been implicated in the disparity of BC mortality rates among AA women. As an example, AA women have a disproportionate incidence of triple negative breast cancer (TNBC), which has a poor prognosis and marginal treatment options. Increasingly, the tumor microenvironment (TME) has gained relevance as it relates to primary tumor progression, metastasis and treatment possibilities. The treatment outcomes or pathological complete response (pCR) in TNBC among AA women are affected by differences in TME. The TME of AA women exhibit several variances in acellular and cellular components associated with pro-tumorigenic effects. For example, increased levels of the adipocyte-related hormone, resistin, the pro-inflammatory cytokine, IL-6, and the CC chemokine, CCL2, within the TME of AA women gives rise to an increased density of M2 macrophages, also known as tumor-associated macrophages. Elevated levels of vascular endothelial growth factor in the TME of AA women increase the vascular density or vascularity, which facilitate aggressive tumor growth and metastasis. Furthermore, a pro-tumorigenic TME is supported by increased levels of the CXC chemokine, CXCL12 that results in the recruitment of regulatory T lymphocytes (Tregs ). Due to these and other differences in the TME of AA women, precision oncology can target specific aspects of the TME that may contribute to a poorer prognosis. In addition to the discrepancies in the TME, AA women face socio-economic barriers that limit their ability to access state-of-the-art, novel therapies against metastatic TNBC. In this review, we will provide a brief overview of the tumor immune microenvironment, immune-based treatment options for TNBC and their potential to decrease health disparities due to ethnicity.

Advances in the Development of Dual-Drug Antibody Drug Conjugates

Antibody drug conjugates (ADCs) are touted for their ability to site selectively deliver a small molecule chemotherapeutic directly to a tumor cell, bypassing off target side effects from systemic circulation. To date, twelve ADCs have been FDA approved in the United States and >200 more in the clinical pipeline. While ADCs have proven successful in both solid and hematological cancers, resistance and tumor heterogeneity are major causes of failure clinically.[1] Tumor heterogeneity is known to lead to recurrence, metastasis, and acquired resistance to ADCs and other therapeutic strategies. Heterogenous tumors with differential drug sensitivities result in aggressive tumor growth, high relapse rates, and poor survival. To combat these challenges, the majority of chemotherapeutic regimens consist of a combination of drugs. Co-delivery of small molecules can overcome resistance, generate additive or synergistic effects, and enhance therapeutic efficacy. Emergence of tumors refractory to current therapies has given impetus to the evaluation of new ADC formats. This challenge has led to the exploration of dual-drug ADCs capable of delivering two mechanistically distinct payloads simultaneously. Strategies for the construction of dual-drug ADCs involve attachment of both drugs to one linker or through the use of two different conjugation sites on the antibody. Herein, we will review the synthesis and evaluation of the dual-drug ADCs reported to date. We focus on ADCs constructed by conjugation of linkers directly to antibody scaffolds and not to other formats or targeting molecules.

S1PR1/S1PR3-YAP signaling and S1P-ALOX15 signaling contribute to an aggressive behavior in obesity-lymphoma

BackgroundExcess body weight has been found to associate with an increased risk of lymphomas and some metabolic pathways are currently recognized in lymphomagenesis. Bioactive lipid metabolites such as sphingosine-1-phosphate (S1P) have been proposed to play an important role linking obesity and lymphomas. However, the underlying mechanism(s) of S1P signaling in obesity-lymphomagenesis have not been well addressed.MethodsThe gene expression of sphingosine kinase (SPHK), lymphoma prognosis, and S1P production were analyzed using Gene Expression Omnibus (GEO) and human lymphoma tissue array. Obesity-lymphoma mouse models and lymphoma cell lines were used to investigate the S1P/SPHK-YAP axis contributing to obesity-lymphomagenesis. By using the mouse models and a monocyte cell line, S1P-mediated polarization of macrophages in the tumor microenvironment were investigated.ResultsIn human study, up-regulated S1P/SPHK1 was found in human lymphomas, while obesity negatively impacted progression-free survival and overall survival in lymphoma patients. In animal study, obesity-lymphoma mice showed an aggressive tumor growth pattern. Both in vivo and in vitro data suggested the existence of S1P-YAP axis in lymphoma cells, while the S1P-ALOX15 signaling mediated macrophage polarization towards TAMs exacerbated the lymphomagenesis. In addition, treatment with resveratrol in obesity-lymphoma mice showed profound effects of anti-lymphomagenesis, via down-regulating S1P-YAP axis and modulating polarization of macrophages.ConclusionS1P/S1PR initiated the feedback loops, whereby S1P-S1PR1/S1PR3-YAP signaling mediated lymphomagenesis contributing to tumor aggressive growth, while S1P-ALOX15 signaling mediated TAMs contributing to immunosuppressive microenvironment in obesity-lymphoma. S1P-targeted therapy could be potentially effective and immune-enhancive against obesity-lymphomagenesis.Graphical

GammaTile® (GT) as a brachytherapy platform for rapidly growing brain metastasis.

A subset of brain metastasis (BM) shows rapid recurrence post-initial resection or aggressive tumor growth between interval scans. Here we provide a pilot experience in the treatment of these BM with GammaTile® (GT), a collagen tile-embedded Cesium 131 (131Cs) brachytherapy platform. We identified ten consecutive patients (2019-2023) with BM that showed either (1) symptomatic recurrence while awaiting post-resection radiosurgery or (2) enlarged by >25% of tumor volume on serial imaging and underwent surgical resection followed by GT placement. Procedural complication, 30-day readmission, local control, and overall survival were assessed. For this cohort of ten BM patients, 3 patients suffered tumor progression while awaiting radiosurgery and 7 showed >25% tumor growth prior to surgery and GT placement. There were no procedural complications or 30-day mortality. All patients were discharged home, with a median hospital stay of 2 days (range: 1-9 days). 4/10 patients experienced symptomatic improvement while the remaining patients showed stable neurologic conditions. With a median follow-up of 186 days (6.2 months, range: 69-452 days), no local recurrence was detected. The median overall survival (mOS) for the newly diagnosed BM was 265 days from the time of GT placement. No patients suffered from adverse radiation effects. Our pilot experience suggests that GT offers favorable local control and safety profile in patients suffering from brain metastases that exhibit aggressive growth patterns and support the future investigation of this treatment paradigm.

Abstract A084: Oncogenic role for upregulated lymphoblastic leukemia derived sequence-1 in the progression of ovarian cancer and its metastasis

Abstract In 2022, about 19,880 women will be newly diagnosed with ovarian cancer (OC), out of which around 12,180 will die. Epithelial Ovarian Cancer (EOC) represents approximately 95% of all OC and is the fifth leading cause of cancer-related deaths in women (deadliest among all gynecological malignancies). Standard treatment for EOC includes surgery, platinum-based chemotherapy, and radiation therapy. Despite these advancements (more than 90% of patients relapse), not all racial and ethnic groups have benefited equally. For instance, the five-year survival rate for OC grew from 33% to 48% among non-Hispanic White women but declined from 44% to 41% among African American (AA) women. The higher tendency to develop chemoresistance in AA women further contributes to their worst survival. No precise mechanisms that contribute to OC racial health disparity have been explained. We aim to delineate the genetic and molecular mechanisms responsible for driving aggressive tumor growth in AA women that disproportionately affect outcomes. This would aid in developing precision therapeutics and eliminate racial disparity in OC outcomes. Analysis of the TCGA data for OC revealed that the Lymphoblastic Leukemia-Derived Sequence 1 (LYL1) gene is one of the frequently amplified genes in OC. About 12% of all the OC shows LYL1 gene amplification. Interestingly, analysis based on ethnicity revealed a remarkable 36% of LYL1 amplification in AA EOC patients, which is one of the top genes after MYC. Importantly, EOC patients with high levels of LYL1 expression (n=202) had a lower survival rate (p=0.003) than those with low levels of LYL1 expression (n=1640). To further confirm the oncogenic functions of LYL1, we analyzed LYL1 expression in EOC cell lines compared to normal fallopian tube epithelial cells (FTECs) and its contribution to OC cell growth and metastatic potential. As expected, LYL1 is upregulated in most of the EOC cell lines compared to normal FTECs, and siRNA-mediated downregulation of LYL1 in EOC cell lines decreased their clonogenic, migration and invasion potential. Conversely, ectopic expression of LYL1 in an EOC cell line expressing low or undetectable levels of LYL1 exhibited increased potential for migration, invasion, and ability to form tumor spheroids. To validate these in vitro studies, we implanted the OVCAR8 cell xenografts transuded with lentivirus-mediated non-targeting shRNAs or shLYL1 into immunocompromised mice. Consistent with the in vitro data, LYL1 expressing OVCAR8 cell xenograft tumors grew aggressively with widespread metastasis in the peritoneum and distant organs. In contrast, LYL1 knockdown tumor xenografts grew slow and showed decreased metastasis. Together, our results show that upregulated or amplified LYL1 in EOC promotes aggressive tumor growth and may contribute to racial disparity in EOC, as this gene is amplified in over 36% of AA OC patients compared to 12% of White patients. Additionally, our studies suggest that LYL1 could be a poor prognostic marker for OC and racial disparities in OC outcomes. Citation Format: Naresh Sah, Swetha Peddibhotla, Bayley Richardson, Pamela Luna, Neel A. Bansal, Chinnadurai Mani, Mark Reedy, Komaraiah Palle. Oncogenic role for upregulated lymphoblastic leukemia derived sequence-1 in the progression of ovarian cancer and its metastasis [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A084.

Association between serum levels of 12 different cytokines and short-term efficacy of anti-PD-1 monoclonal antibody combined with chemotherapy in advanced gastric cancer.

Gastric cancer (GC) is characterized by aggressive tumor growth and poor prognosis. The benefits of targeted anti-programmed death receptor 1 (PD-1) monoclonal antibody combined with chemotherapy have not yet been characterized. The tumor microenvironment and circulating factors have garnered interest as possible predictors of response and prognosis. The aim of this study was to evaluate whether cytokine levels in the serum of patients were related to tumor response to anti-PD-1 monoclonal antibody combined with chemotherapy and survival in advanced GC. Preoperative serum samples were collected from patients with GC (n=52) and healthy individuals (n=31). The levels of 12 different cytokines were measured using a multiple microsphere flow immunofluorescence assay. The association between cytokine levels and clinical response was analyzed using nonparametric Wilcoxon matched-pair ranked tests. Progression-free survival (PFS) time for all patients was recorded via evaluation of imaging results and follow-up via telephone. Kaplan-Meier and log-rank tests were used to plot survival curves. The levels of interleukin (IL)-6, IL-1β, interferon (IFN)-γ, IL-17, and IL-12p70 in the control group were significantly lower than those in the GC group (p=0.0002, p=0.0065, p=0.0003, p=0.0303, and p=0.0295, respectively). The level of IL-4 was significantly higher in healthy individuals than that in patients with GC (p=0.0201). The cytokine levels in the good responder group were higher than those in the poor responder group before therapy. Patients treated with immunochemotherapy showed an overall reduction in all cytokine levels after treatment initiation. A high baseline level of IFN-γ was associated with a better prognosis. However, high IL-6 levels in patients after two cycles of immunochemotherapy indicated resistance. High IL-4 levels in patients treated with four cycles of immunochemotherapy were associated with better PFS. Our study suggests that low levels of IFN-γ before immune checkpoint inhibitor treatment may be useful for the detection of a poor immunological status. Hence, a reduction in IL-6 levels is predictive of a longer PFS, and increased IL-4 levels are predictive of a good response. IL-4 and IL-6 may, therefore, serve as promising circulating predictive biomarkers for patients who can benefit from anti-PD-1 monoclonal antibodies administered in combination with chemotherapy.

Abstract C027: miRNA-214-5P regulates RAD51, a biomarker for aggressive disease and racial disparities in triple-negative breast cancer

Abstract Breast cancer (BC) is one of the deadliest cancers in women. Among various subtypes, triple-negative breast cancer (TNBC) is the most aggressive and hard to treat subtype of breast cancer because it is highly metastatic and lacks targeted therapies. The death rate from BC is higher among African American (AA) women than among women of other races and ethnicities. The higher incidences of TNBCs and their aggressive growth in young AA women contributing to higher death rates indicate a biological basis for this difference. Thus, it is imperative to understand the molecular mechanisms that contribute to aggressive tumor growth in AA women, identify biomarkers to select patients who will respond to existing therapies, and develop effective therapeutics to reduce this disparity. Our previous findings showed that the DNA repair protein, RAD51, is overexpressed in AA TNBC patients and correlates with a poor prognosis relative to European American (EA) TNBC patients. However, the exact mechanism behind the regulation of RAD51 has not been identified. Our miRNA seq analysis shows a list of downregulated miRNAs in AA TNBC cell lines compared to EA TNBC cell lines. Interestingly, the miRDB-MicroRNA Target Prediction Database predicted that miR-214-5P has the seed sequence to bind and degrade RAD51 mRNA. Analysis of the TCGA database by UALCAN portal also shows a decreased expression of miR-214-5P in AA TNBC patients compared to EA TNBC patients. Treating the AA TNBC cell lines with miR-214-5P mimic downregulates RAD51 expression in a cell cycle-independent manner and also induces HR-deficiency as measured by Dr-GFP assay. Based on these results, we designed a synergistic lethality-based combination of miR-214-5P and Olaparib in TNBC cells. Data from our preclinical evaluations show miR-214-5P and Olaparib cause increased DNA strand breaks, and synergistic TNBC cell lethality compared to individual treatments. Together, our data indicate that miR-214-5P regulates RAD51 and either of these genes could be biomarkers for aggressive TNBC and racial disparity in BC therapeutic outcomes. Citation Format: Ganesh Acharya, Chinnadurai Mani, Upender Manne, Komaraiah Palle. miRNA-214-5P regulates RAD51, a biomarker for aggressive disease and racial disparities in triple-negative breast cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C027.

Abstract B067: Genetic and pharmacological inhibition of phospholipase D1 reduces pancreatic carcinogenesis in mice

Abstract Pancreatic ductal adenocarcinoma (PDA) is a leading cause of cancer-related death in the United States and is characterized by its sudden manifestation, poor prognosis, high incidence of metastasis, and aggressive tumor growth. Phospholipase D1 (PLD1) is a lipid-signaling enzyme that plays a role in signaling pathways that lead to cell proliferation, survival/apoptosis as well as downstream oncogenic transformation involved in cancer progression. PLD1 activity has been shown to promote tumor progression in other cancer types, however, the role and mechanisms of PLD1 in pancreatic cancer is not yet understood. The objective of this study is to determine the role of PLD1 regulation in pancreatic carcinogenesis through genetic-ablation and pharmacological inhibition of PLD1. In this study, we crossed the LSL-KrasG12D/+;Ptf1Cre/+ (KC) mice into PLD1 knockout (Pld1-/-) mice to generate KC; Pld1-/- mice in the C57BL/6 background to examine whether PLD1 is required for pancreatic tumorigenesis. Cohorts of male and female KC and KC; Pld1-/- mice, (8-12 mice per group), were euthanized at 4 and 8 months old and the pancreas was carefully resected. At 8 months of age, there was a significant difference (p<0.05) in the pancreas weight at sacrifice between the KC mice and the KC; Pld1-/- mice in a sex-dependent manner. This was due, in part, by a reduction in tumor proliferation as determined by Ki67 expression. To assess whether pharmacological inhibition of PLD could prevent pancreatic carcinogenesis in KC mice, cohorts of 9-12 male 7-month-old KC mice received daily intraperitoneal injections of a small molecule inhibitor of PLD (FIPI) at a dosage of 3 mg/kg of body weight for a duration of 5 weeks. The mice were euthanized at 8.5 months of age and the pancreas was carefully resected. Histological analysis indicated that FIPI-treated KC mice displayed less acinar cell loss compared to vehicle-treated KC controls. Moreover, treatment with FIPI significantly reduced cell proliferation (p<0.01) in KC mice in comparison to vehicle-treated KC mice. In summary, these results indicate that PLD1 plays a critical role in pancreatic carcinogenesis and may represent a novel therapeutic target. Additional studies are underway to explore the cellular mechanisms of how PLD1 inhibition modulates pancreatic carcinogenesis. Citation Format: Hala A. Addassi, Gerardo G. Mackenzie. Genetic and pharmacological inhibition of phospholipase D1 reduces pancreatic carcinogenesis in mice [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B067.

Tumor growth inhibition and immune system activation following treatment with thorium-227 conjugates and PD-1 check-point inhibition in the MC-38 murine model

Targeted thorium-227 conjugates comprise the combination of a monoclonal antibody with specificity for a tumor cell antigen and a 3,2-HOPO chelator enabling complexation of thorium-227 (Th-227). The radiolabeled conjugate functions as an effective delivery system of alpha-particle radiation to the surface of the tumor cell inducing difficult to repair complex DNA damage and cell death. In addition, the mechanism of action of targeted alpha therapy (TAT) appears to involve a significant component linked to stimulation of the immune system. We report herein evidence of immune activation and long-lasting immune protection of a TAT in a syngeneic model using the MC-38 murine cell line. Firstly, MC-38 cells were irradiated ex vivo with the thorium labeled antibody before subcutaneous implantation into mice. These mice were then rechallenged with MC-38 cells contra-laterally. In the group receiving irradiated cells, 9 out of 10 animals had no measurable tumor growth compared to aggressive tumor growth in the control group. Secondly, in an efficacy study, 500 kBq/kg of thorium labeled antibody alone or in combination with PD-1 checkpoint inhibitor gave statistically significant tumor growth inhibition compared to vehicle control. Animals with no measurable tumors were once again rechallenged contra-laterally with MC-38 cells. The re-growth of tumors was significantly delayed (approx. 60 days) in the treatment group compared to age-matched controls (approx. 30 days) in the monotherapy group. Interestingly, in the TAT/ PD-1 combination group no re-growth was observed demonstrating the potential of combining a TAT with checkpoint inhibition therapy. Finally, tumors were excised from treated mice and analyzed by flow cytometry and immunohistochemistry (IHC). Analysis revealed significant infiltration of CD8+ T-cells and mature dendritic cells compared to vehicle controls. Together these results indicated that an ongoing immune response from treatment with alpha radiation could be enhanced by check-point inhibition.