bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 237: Evaluation of cell membrane lipid-extracted nanoliposomes (CLENs) as a potential drug platform for targeting multiple myeloma
Authors: Tran Christina T. , Park Eden , Rodriguez-Flores Pedro L. , Campbell Robert B.
Published: June 2022

Background: Multiple myeloma (MM) is a plasma cell malignancy that primarily affects the elderly. MM is the second most common hematologic cancer with approximately 25,000-30,000 new cases diagnosed annually in the United States. A challenge associated with MM therapy is unwanted drug uptake by normal healthy cells. Nanoparticles provide a promising outlook to mitigate these issues, offering more selective drug uptake, while minimizing toxicity associated with treatment. The objective of this study was to develop, optimize and evaluate physicochemical and In vitro properties of MM-CLENs in comparison to conventional liposomes and relevant cell controls.

Methods: RPMI-8226 MM cell line was selected cellular model for MM disease. RPMI-8226 cells were cultured and expanded In vitro for cellular extraction purposes. Lipid extraction (LE) procedures were performed to generate the RPMI-8226 LE ingredient. Other lipid ingredients used in preparation of RPMI-8226 CLENs included DOPC, cholesterol, and DPPE-Rhodamine (fluorescence indicator). Phase I included physicochemical characterization and cellular uptake studies of CLENs preparations with varying concentration of RPMI-8226-LE (0 to 40 mol%) content. Phase II began with the optimized concentration of LE determined in Phase I, along with varying concentrations of cholesterol (0 to 40 mol %) content for the nano-targeting studies in vitro. The control cell lines represent other hematological diseases, K-562-GFP (chronic myeloid leukemia), CCRF-CEM (acute lymphoblastic leukemia), and U937 (lymphoma).

Results: Phase I and II physicochemical characterization and In vitro studies, revealed that the optimized preparation of RPMI-8226-CLENs consisted of 5% LE, and 5% cholesterol content. Optimal preparation consisted of DOPC: Cholesterol: LE (90:5:5) with an average values for particle size and zeta potential of 178±38 nm (n=4), and -14±4 mV (n=4), respectively. The inclusion of LE in conventional DOPC:cholesterol liposomes resulted in significantly improved uptake of the CLENs by RPMI-8226 cells in vitro (P< 0.001). However, preliminary selectivity studies suggest no significant difference in the uptake of RPMI-8226-CLENs by RPMI-8226 when compared to control cells (K-562-GFP, CCRF-CEM and U937).

Conclusion: Studies involving RPMI-8226 CLENs are currently underway. Such studies include target selectivity, and influence of additional components of CLENs as a function of the microenvironment.

Citation Format: Christina T. Tran, Eden Park, Pedro L. Rodriguez-Flores, Robert B. Campbell. Evaluation of cell membrane lipid-extracted nanoliposomes (CLENs) as a potential drug platform for targeting multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 237.
Abstract 2377: Impairment of p53-activating pathways accelerates liver cancer initiation partially through MTHFD1L-mediated 1C metabolism
Authors: Zhang Yiwei , Zha Yitian , Lu Hua
Published: June 2022

Hepatocellular carcinoma (HCC) takes several decades to develop from premalignant lesions in chronically damaged livers and is often detected at a later stage; thus, prevention is of particular importance. HCC originates from differentiated hepatocytes. Previous studies have shown that inhibition of the tumor suppressor p53 is required for the differentiated hepatocytes to acquire the features that are needed for their conversion to HCC progenitor cells (HcPCs). However, the initiation of p53 response to de-differentiation of damaged hepatocytes is largely unclear. p53 is tightly controlled by MDM2 and MDMX. We and others have previously demonstrated that multiple stresses can trigger the binding of 14-3-3 to phosphorylated MDMX, or ribosomal proteins L5 and L11 to MDM2, to further disrupt the MDMX- or MDM2-p53 association and lead to p53 activation. This was validated by two animal models, MDM2C305F and MDMX3SA knock-in mice that are defective in 14-3-3-MDMX and L5/L11-MDM2 interactions, respectively. By crossing these two mouse lines, we generated a double knock-in (DKI) mouse line. With this line, we found that carcinogen DEN-induced HCC occurrence is significantly higher in MDM2C305F-MDMX3SA -DKI mice, and HcPCs isolated from DEN-treated DKI mice display significantly more stem cell- and malignancy- properties and higher tumorigenesis when introduced into mouse livers. Furthermore, we found that MTHFD1L, a monofunctional tetrahydrofolate synthase required for mitochondrial 1-carbon (1C) metabolism, is transcriptionally suppressed by p53 and exerts a critical effect on the accelerated HCC initiation in DKI mice. Taken together, these results demonstrate a crucial role of these two p53-activating pathways in preventing HCC initiation by controlling the mitochondrial 1C- metabolism.

Citation Format: Yiwei Zhang, Yitian Zha, Hua Lu. Impairment of p53-activating pathways accelerates liver cancer initiation partially through MTHFD1L-mediated 1C metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2377.
Abstract 2386: Investigating cyclopamine tartrate as a potential suppressor of lung adenocarcinoma and squamous cell carcinoma
Authors: Berisha Eranda , Ashrafi Adnin , Castro Maria Del Carmen Chacon , Zhang Li
Published: June 2022

Lung cancer is the leading cause of death by cancer, and non-small cell lung cancer comprises of 85-90% of lung cancer cases. Previous studies have demonstrated the importance of heme in mitochondrial metabolism and OXPHOS. Heme is an essential molecule in the oxidative phosphorylation pathway especially since Complex II-IV all contain heme. During lung tumorigenesis there is a marked increase in heme synthesis and uptake; other protein and enzymes relating to heme were also upregulated. Heme-sequestration proteins (HeSPs) have shown high effectiveness in suppressing the development and progression of lung adenocarcinoma and squamous cell carcinoma, therefore targeting heme using CycT should yield similar results. Previous studies have indicated that cyclopamine tartrate (CycT) can effectively arrest lung tumor growth and progression. CycT does this by inhibiting the levels of proteins and enzymes involved in heme uptake, synthesis, as well as reducing heme synthesis and degradation in adenocarcinoma. We therefore aim to examine the effectiveness of CycT in both, adenocarcinoma and squamous cell carcinoma.In vitro experiments using NSCLC adenocarcinoma cell line A549 and squamous cell carcinoma HCC15 are in progression with initial results supporting the hypothesis. Cell proliferation assays have shown a marked decrease in CycT-treated groups in both cell lines. Based on colony formation assays, results indicate that CycT inhibits colony formation. Heme synthesis and degradation have also shown the effectiveness of CycT in suppressing tumorigenesis in both cell types. Oxygen consumption rates of both cell types were examined with CycT inhibiting oxygen consumption. Overall, in vitro experiments support the idea that CycT can suppress or arrest lung adenocarcinoma and squamous cell carcinoma.

Citation Format: Eranda Berisha, Adnin Ashrafi, Maria Del Carmen Chacon Castro, Li Zhang. Investigating cyclopamine tartrate as a potential suppressor of lung adenocarcinoma and squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2386.
Abstract 2394: Discovery of a sex-specific metabolic phenotype in KRAS mutant colorectal cancer
Authors: Yan Hong , Cai Yuping , Shen Xinyi , Jain Abhishek , Paty Philip B , Bosenberg Marcus , Khan Sajid A , Johnson Caroline
Published: June 2022

KRAS mutations occur in 35-45% of all colorectal cancer (CRC) cases and are notoriously difficult to treat. It is known that anomalous metabolism is a hallmark for cancer, however the relationship between mutant KRAS and tumor metabolism in CRCs has not been fully explored. We recently showed sex-disparities in metabolism by primary tumor location, indicating the pressing need to investigate the role of biological sex in CRC. Our aim was therefore to determine whether mutant KRAS alters metabolism in a sex-specific manner. We performed non-targeted metabolomics and targeted metabolomics on stage I-III chemotherapy-naive KRAS-mutant and wild type CRC patient tumor tissues (n=197) using ultra-high-performance liquid chromatography (UHPLC) coupled to quadrupole Time-of-Flight Mass Spectrometry (QTOF-MS), and tandem MS (MS/MS). Our results showed that tumors from male patients were cystine(cysteine)-dependent, with altered glutathione (GSH) biosynthesis, transsulfuration activity, methionine metabolism, utilization of lipoxygenase pathways. Simultaneously, we performed comprehensive bioinformatic analysis of gene expression data from the Gene Expression Omnibus to further validate mutant KRAS effects on CRCs by sex. Our findings demonstrated differential expression to CBS, GPX4, GSS, ACSL4, ATF4, FTH1 and EGPR genes that are all involved in regulation of these metabolic pathways and indicate a role for ferroptosis. This data therefore validated our findings of decreased transsulfuration activity (regulated by CBS) and decreased GSH biosynthesis (regulated by GSS) in male patients; this finding was not seen in female patients. To further examine differences in ferroptosis by sex, it was seen that GPX4 was differentially expressed between KRAS mutant and wild type (p<0.05) in males, but not in females. Meanwhile, ACSL4, which appears to be a predictive marker for ferroptosis sensitivity, showed upregulated expression in males with mutant KRAS (p<0.00001). Further, KRAS mutant type linked to a poor 5-year overall survival only in male using Kaplan-Meier survival analysis. This is the first report to show unique sex-specific metabolic signatures by KRAS status in CRC patients indicating a role of decreased ferroptosis in males, thus a novel avenue for therapeutic approaches.

Citation Format: Hong Yan, Yuping Cai, Xinyi Shen, Abhishek Jain, Philip B Paty, Marcus Bosenberg, Sajid A Khan, Caroline Johnson. Discovery of a sex-specific metabolic phenotype in KRAS mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2394.
Abstract 2403: Regulation of FAK phosphorylation by serine/threonine protein phosphatases in cancer cells
Authors: Li Lin , Liu Lie , Huang Shile
Published: June 2022

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, is activated by growth factors and integrin clustering, promoting cell migration, a critical step for cancer metastasis. The activity of FAK is closely associated with the phosphorylation of diverse residues, which are mediated by various protein tyrosine kinases and phosphatases. Previous studies have demonstrated that serine/threonine protein phosphatase 5 (PP5) promotes cell migration, while protein phosphatase 2A (PP2A) inhibits cell migration. However, whether PP5 and PP2A regulate cell migration by mediating FAK phosphorylation is poorly understood. In this study, we found that overexpression of PP5 decreased FAK phosphorylation at S722, and knockdown of PP5 increased p-FAK (S722) in a spectrum of tumor cell lines. Our co-immunoprecipitation (co-IP) results showed an interaction between PP5 and FAK. In addition, we observed that overexpression of PP5 induced S178 phosphorylation of paxillin, an FAK interacting protein. However, there was no physical interaction between PP5 and paxillin, indicating an indirect regulation. In addition, knockdown of PP2A enhanced the phosphorylation of FAK and conferred resistance to rapamycin inhibition of IGF-I-stimulated phosphorylation of FAK. To our surprise, by co-IP, we failed to detect any direct interaction between FAK and PP2A. Further research will determine how PP5 and PP2A regulate cell migration through FAK and paxillin.

Acknowledgements This work was supported by the Feist-Weiller Cancer Center (FWCC) Bridge Award (S.H.), Shreveport, LA, USA

Citation Format: Lin Li, Lie Liu, Shile Huang. Regulation of FAK phosphorylation by serine/threonine protein phosphatases in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2403.
Abstract 2412: Emergence of resistance to MTI-101 selects for favorable MET phenotype in lung cancer
Authors: Jones Clark , Hazlehurst Lori
Published: June 2022

Lung cancer remains the most-deadly of all cancers, and thus clinical evidence indicates novel treatment strategies are needed to improve patient outcomes. Our laboratory has been developing a novel cyclic peptide coined MTI-101, which induces cell death via calcium overload, a finding dependent on the TRPC1/TRPC5/TRPC4 complex. To better understand determinants of sensitivity and resistance to MTI-101, we developed drug resistant isogenic lung cancer cell lines. Two isogenic drug resistant cell lines were generated with chronic exposure to MTI-101 in PC-9 (NSCLC EGFR Driven) and H446 (SCLC PTEN Deleted and c-MYC amplified). Drug resistance was confirmed in both cell lines with over two-fold increases in IC50 values upon treatment with MTI-101 in MTT assays. Interestingly, pushing the resistant levels beyond a two-fold increase was not obtained as cultures did not sustain growth with increased selection pressure. Using the isogenic systems coupled with RNA-SEQ, the top enriched GSEA hallmark was downregulation of genes that contribute to EMT in both MTI-101 resistant lines. The RNA-SEQ data correlated with changes in the phenotype which included a significant decreased invasion in Matrigel and changes of MET markers (E-Cadherin, Vimentin) at the protein level. Furthermore, in the EGFR driven PC-9 cell line, selection for resistance towards MTI-101 resulted in collateral sensitivity to EGFR inhibitors. To determine the stability of the MET phenotype, cells were removed from drug, then tested for invasion and drug resistance every month. Our data indicated that the MET phenotype and genotype was stable for a minimum of six months. RNA-SEQ data continued to demonstrate decreased expression of EMT in cells removed from drug compared to the parental cell line. MTI-101 treatment in PC-9 and H446 cells showed synergistic activity with standard of cares Erlotinib, Osimertinib, and Cisplatin when used in combination on the wildtype cells as well. Finally, in vivo data indicates that MTI-101 treatment selects for increased E-Cadherin and decreased Vimentin. Similar to the cell line model, this finding correlated with decreased incidence of bone metastasis in the PC-9 in vivo model. Together our data indicate that MTI-101 treatment could be used as a tool to delay the emergence of resistance and potentially inhibit or delay the emergence of metastatic disease for the treatment of lung cancer.

Citation Format: Clark Jones, Lori Hazlehurst. Emergence of resistance to MTI-101 selects for favorable MET phenotype in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2412.
Abstract 2414: Retinoic acid induced 14 drives pancreatic cancer progression and metastasis
Authors: Runa Farhana , Tomaneng Luke , Adamian Yvess , Cox Nathan , Aquino Albert-Fred , Wallace Matthew , Gonzalez Carolina , Bhakta Kishan , Hoover Malachia , Wolfenden Laurelin , Humphries Jonathan D. , Humphries Martin J. , Boyce Michael , Kelber Jonathan A.
Published: June 2022

Pancreatic ductal adenocarcinoma (PDAC) is associated with very poor outcomes - fewer than 10 percent of patients survive beyond five years after diagnosis. The primary hurdles facing PDAC patients and clinicians are early dissemination, a lack of therapeutic targets and desmoplasia that renders the primary tumor refractory to chemotherapy. In this regard, we have previously reported that pseudopodium-enriched atypical kinase 1 (PEAK1) and integrin α1 (ITGA1) mediate gemcitabine resistance and metastasis in PDAC. To identify new mechanisms of PDAC progression, we mined the Cancer BioPortal and Human Cell Map BioID databases for additional pseudopodium-enriched (PDE) proteins that predict poor patient outcomes, correlate with PEAK1 and ITGA1 expression in PDAC, and interact with PEAK1 and ITGA1. Here, we identify Retinoic Acid Induced 14 (RAI14, Ankycorbin or NORPEG) as a new candidate driver of PDAC that is a constituent of the KRasG12D PDAC cell autonomous phosphoproteome, localizes to cytoskeleton/adhesion domains in PDAC cells and correlates in expression with ITGA1-binding collagens in PDAC. Knockdown or knockout of RAI14 in KRas mutant PDAC cells impaired adhesion-dependent proliferation/survival in vitro and tumor growth and metastasis in vivo. Single cell cyclic immunofluorescence (CycIF) further revealed that RAI14 supports a subpopulation of PDAC cells positive for proliferation, epithelial to mesenchymal transition (EMT) and antiapoptotic programs. By using a RAI14-focused bioinformatics pipeline in combination with proteomic and immunofluorescence data on the composition of ITGA1-dependent adhesion complexes in PDAC cells, we identified Polo-Like Kinase 1 (PLK1) as a candidate that may control RAI14 function and adhesion-regulated mitosis during PDAC progression. Notably, the potency of volasertib, a PLK1-specific inhibitor, was reduced in RAI14 knockout cells, supporting a model in which RAI14 mediates adhesion-dependent PLK1 functions in PDAC. Taken together, these studies uncover a mechanism for RAI14-driven PDAC progression and the development of strategies to increase chemotherapy sensitivity, reduce primary/metastatic tumor burden and improve patient outcomes.

Citation Format: Farhana Runa, Luke Tomaneng, Yvess Adamian, Nathan Cox, Albert-Fred Aquino, Matthew Wallace, Carolina Gonzalez, Kishan Bhakta, Malachia Hoover, Laurelin Wolfenden, Jonathan D. Humphries, Martin J. Humphries, Michael Boyce, Jonathan A. Kelber. Retinoic acid induced 14 drives pancreatic cancer progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2414.
Abstract 244: Limiting metastatic breast cancer progression by targeting MK2 stromal signaling
Authors: Oliveira Renata Ramalho , Faget Douglas V. , Luo Xianmin , Ye Jiayu , Monahan Joseph B. , Stewart Sheila A.
Published: June 2022

Breast cancer is now the most common cancer among women in the US and metastasis to distant organs is the major cause of death in these patients. Among metastatic sites, bone is the most frequent destination of breast cancer metastasis and once the disease is found in the bone, it is often refractory to standard therapy. In addition, bone metastasis is associated with significant skeletal related events that greatly impact quality of life. Thus, there is a significant need to develop novel therapies that extend survival and improve quality of life. Previously we found that MK2 inhibition (MK2i) limits metastatic growth by targeting the tumor stromal compartment. To determine which stromal cells are targeted by MK2i we isolated stromal cells from within a metastatic bone lesion and carried out single cell RNA sequencing (scRNA-Seq). Using this approach, we found numerous gene expression changes in both the immune (CD45+) and non-immune (CD45-) populations upon MK2i. Indeed, we found significant numbers of p16+ senescent fibroblasts were present inside the metastatic lesion. We also found vascular cancer-associated fibroblasts (vCAF) and matrix CAFs (mCAF) inside the metastatic lesion suggesting that CAFs are not limited to primary disease. Numerous changes in gene expression in CD45+ cells were also observed, and these changes are being used to identify viable immunotherapies to deploy in our model. Finally, we are using conditional MK2 mice to establish the cell type targeted by MK2i. Our data demonstrate that MK2i is as effective as chemotherapy at limiting metastatic progression and our approach to leverage the scRNA-Seq data may allow us to optimize MK2i therapy to improve patient outcomes and quality of life.

Citation Format: Renata Ramalho Oliveira, Douglas V. Faget, Xianmin Luo, Jiayu Ye, Joseph B. Monahan, Sheila A. Stewart. Limiting metastatic breast cancer progression by targeting MK2 stromal signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 244.
Abstract 2442: Seminal fluid enhances cervical squamous carcinoma cell proliferation, epithelial mesenchymal transition (EMT), migration & invasion
Authors: Mkwanazi Nonkululeko , Leaner Virna D. , Katz Arieh A.
Published: June 2022

Cervical cancer is the leading cause of cancer related deaths & the second most common cancer amongst South African women. Though there are various risk factors, the most common risk factor for cervical cancer development is sexual transmission & persistent infection with high-risk Human Papillomavirus (HPV). Although most HPV infections are cleared naturally in women, persistent infection with high-risk HPV types promotes neoplastic transformation & tumorigenesis. Furthermore, exposure of neoplastic cervical epithelial cells to inflammatory mediators may drive the progression of cervical cancer. Seminal fluid (SF), a biological fluid rich in inflammatory mediators & growth factors, has been implicated in cervical cancer progression. SF has been shown to induce the expression of proinflammatory mediators which promotes cell proliferation in culture & growth of explants in mice injected with HeLa cervical adenocarcinoma cells. Since majority of cervical cancer cases are squamous cell carcinoma, in this study, we examined the effect of SF on cell proliferation, EMT, cell migration & invasion employing SiHa and Me180 squamous cell carcinoma cell lines. Our results showed that SF significantly enhanced cell proliferation in both cell lines. Using Confocal microscopy and phalloidin staining we further showed that SF caused morphological changes and stress fibre formation. RT-qPCR showed that SF up regulated EMT transcription factors Snail, ZEB1 & TWIST mRNA expression. EMT induction was further confirmed by the increase of N-cadherin & a subsequent decrease in E-cadherin protein expression. Additionally, we showed that the induction of EMT transcription factors by SF occurs via the EGF/ERK/COX pathway. To investigate the effect of SF on migration and invasion, transwell migration assays were used. Results showed that SF significantly enhanced cell migration & invasion of the SiHa and Me180 cells. Our results show that SF plays a role in enhancing the migratory & invasive potential of squamous cell carcinoma cells & that this associates with increased expression of EMT transcription factors. These findings together implicate SF as a possible factor that may promote cervical cancer progression.

Citation Format: Nonkululeko Mkwanazi, Virna D. Leaner, Arieh A. Katz. Seminal fluid enhances cervical squamous carcinoma cell proliferation, epithelial mesenchymal transition (EMT), migration & invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2442.
Abstract 2444: The integrin alpha10beta1 antibody TARG10 reduces tumor growth and metastasis of triple negative breast cancer
Authors: Masoumi Katarzyna , Huang Xiaoli , Mirkov Anna , Smedenfors Kristine , Norlén Per , Lundgren-Åkerlund Evy
Published: June 2022

Triple negative breast cancer (TNBC) is an aggressive form of breast cancer that accounts for 10-15% of all breast cancer diagnoses. It is far more aggressive than other breast cancer types due to a higher degree of metastases and frequent relapses following current treatment options. Integrin alpha10beta1 is a collagen binding receptor that has emerged as a promising new cancer target due to its role in tumor cell proliferation and migration. The aim of the present study was to investigate the function blocking activity of the human integrin alpha10beta1 antibody TARG10 on growth and metastasis of TNBC.We have found that integrin alpha10beta1 is highly expressed in TNBC, with minimal expression in healthy tissue, and that high expression is correlated with poor prognosis in TNBC patients. In vitro studies showed significant inhibiting effects of TARG10 on the adhesion, migration, proliferation, and viability of TNBC cells. In an orthotopic xenograft TNBC model, TARG10 confirmed inhibitory effects on both tumor growth and metastasis as demonstrated by In Vivo Imaging System (IVIS-CT). The effect on metastasis was further demonstrated in a tail vein xenograft TNBC model of experimental metastasis. Pharmaceutical evaluation demonstrated excellent developability properties, including low post translational liabilities, high melting point, low aggregation and high yield.In summary, integrin alpha10beta1 represents a promising target for directed antibody-based therapy of TNBC. Our in vitro and in vivo data supports pre-clinical and clinical development of TARG10 as a candidate drug for treatment of patients with triple negative breast cancer.

Citation Format: Katarzyna Masoumi, Xiaoli Huang, Anna Mirkov, Kristine Smedenfors, Per Norlén, Evy Lundgren-Åkerlund. The integrin alpha10beta1 antibody TARG10 reduces tumor growth and metastasis of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2444.
Abstract 246: Application of the 3d fresh patient tumoroid platform 3d-explore to assess the immunomodulatory and phagocytic activity of the receptor tyrosine kinase inhibitor, Sunitinib
Authors: Bunch Brittany , Kodumudi Krithika N. , Ehrhart Jared , Weitzman Matt , MacIntosh Olivia , Sussman Kelly , Altiok Soner
Published: June 2022

Background: The receptor tyrosine kinase inhibitor (TKI) Sunitinib has proven useful in the treatment of a variety of tumors. Sunitinib has shown therapeutic activity in patients with metastatic renal cell carcinoma and currently represents a frontline therapy for this disease. The immune modulatory effect of Sunitinib on immune cells within the tumor microenvironment has been well documented. Here, we developed a novel 3D ex-vivo platform (3D-EXplore) using fresh patient tumor samples with intact stromal components and tumor immune microenvironment to assess the therapeutic efficacy of Sunitinib.

Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids with intact TME measuring 150 µm in size were prepared from fresh tumor samples of renal cell carcinoma using a proprietary technology developed at Nilogen Oncosystems. Tumoroids prepared from each patient’s tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with Sunitinib for 48h to detect treatment-mediated changes in tumor immune cell composition including CD4 and CD8 T-cells, NK cells, and macrophages. Additionally, we analyzed treatment-mediated changes in T-cell activation and phagocytic activity of myeloid cells.

Results: Multiparameter flow analysis revealed that Sunitinib treatment led to an increase in CD8+CD25+ central memory-like phenotype and increased granzyme B expression in CD4 and CD8+ T cells indicating T cell activation. Furthermore, multiparameter flow cytometry analysis showed increased EdU uptake by CD8 and NK cells with sunitinib treatment, while cytokine release assays demonstrated increased IFN-γ and IL-2 production accompanied by downregulation of IL-10 and IL-6 cytokines. We observed Sunitinib mediated phagocytotic activity by tumor resident myeloid macrophages, monocytes, and dendritic cells using a quantitative phagocytosis assay.

Conclusions: These results demonstrate that 3D-EXplore using fresh tumor samples provides a clinically relevant platform to assess drug response and serves as a critical tool for reflecting the true intact tumor microenvironment for novel immune-oncology drug development.

Citation Format: Brittany Bunch, Krithika N. Kodumudi, Jared Ehrhart, Matt Weitzman, Olivia MacIntosh, Kelly Sussman, Soner Altiok. Application of the 3d fresh patient tumoroid platform 3d-explore to assess the immunomodulatory and phagocytic activity of the receptor tyrosine kinase inhibitor, Sunitinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 246.
Abstract 2485: Applications of multimodality ex-vivo tissue imaging to improve breast cancer diagnosis and treatment
Authors: Avila Corazon , Karczmar Gregory , Mustafi Devkumar
Published: June 2022

Breast cancer is the most common malignancy affecting women worldwide. Existing models being used to study breast cancer include the Patient-Derived Tumor Organoid (PDTO) Model and the Patient-Derived Tumor Xenograft (PDTX) Model. Both models involve the surgical removal of a tumor from a patient which then undergoes processing to grow an organoid or injected into mice to test different therapies outside the body. However, these models not only take a considerable amount of time to develop properly, but in many cases aren’t representative of the most aggressive tumor cells present within a heterogeneous tissue sample in order to draw the most clinically accurate conclusions of a tumor's response to therapy. In order to advance clinical research, more efficient culture systems that are faster growing and more representative of biological characteristics in-vivo are needed. Previous work from the Karczmar Lab discovered that high-resolution magnetic resonance imaging (MRI) can detect regions of aggressive cancer in biopsy specimens from mouse models, and give additional information on characteristics including cell density, tissue composition, and cancer stage. With this recent discovery, it is hypothesized that ex-vivo imaging could be used to identify aggressive tumor cells in human heterogeneous breast cancer tissue samples to produce higher quality and faster-growing models for testing new therapies and guiding individual patient therapy for breast cancer. The primary aim for this study was to specifically identify image-based markers for regions of aggressive cancer by comparing ex-vivo high-resolution MRI with histology and immunohistochemistry in 3D. Distant normal (DN) and tumor (Tu) tissue samples from breast cancer patients were obtained and imaged using T2-weighted 3D Rapid Imaging with Refocused Echoes (RARE) and stained using hematoxylin and eosin (H&E), commonly used by pathologists to diagnose cancers. Then, the ex-vivo T2-weighted 3D RARE images were correlated to the histopathological H&E staining and confirmed by breast radiologists and pathologists. Preliminary results revealed that high-resolution MR imaging detects mammary glands, ducts, and white adipose tissue in DN tissue samples and regions of invasive cancer, including ductal carcinoma in-situ (DCIS) and necrosis, in Tu tissue. Based on these results, aggressive cancer regions were proven to be identified using image-based guidance and potentially be extracted using core-needle biopsy to improve PDTO and PDTX models for studying breast cancer and advancing individual patient therapy.

Citation Format: Corazon Avila, Gregory Karczmar, Devkumar Mustafi. Applications of multimodality ex-vivo tissue imaging to improve breast cancer diagnosis and treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2485.
Abstract 2521: Impact of peritoneal lavage on omentum immune cell dynamics and metastasis
Authors: Akasaka Hironari , Naora Honami
Published: June 2022

Peritoneal lavage is a routine procedure in which normal saline solution is administered into the abdominal cavity and the washings are cytologically examined for cancer cells. Peritoneal lavage often strips protective mesothelial linings and can promote formation of peritoneal adhesions. However, the impact of peritoneal lavage on the omentum, which is also exposed to this procedure, is unclear. The omentum is a fatty tissue that suspends from the stomach and contains highly vascularized immune cell structures called milky spots. Because circulating ovarian and gastric cancer cells have strong predilection for implanting in milky spots, it is important to understand the impact of peritoneal lavage on the omentum, as not all patients with these cancers undergo omentectomy. In this study, we administered clinically relevant amounts of normal saline solution to immunecompetent mice to mimic peritoneal lavage, and evaluated the absorption of saline solution and the gross architecture and immune cell composition of the omentum and other peritoneal fat tissues. As compared to other peritoneal fat tissues, the omentum showed the highest capability to absorb saline solution. Following administration of saline, a dramatic expansion in milky spots and increased vessel density were observed in the omentum. Immunophenotyping and functional studies revealed that these cellular changes were due in part to the expansion of a sub-population of peritoneal macrophages. Furthermore, studies using mouse allograft models of ovarian cancer revealed that saline administration stimulated the implantation of circulating cancer cells on to the omentum. Collectively, these findings raise the possibility that peritoneal lavage ‘primes’ the omentum to be conducive for colonization by occult cancer cells that circulate in the peritoneal fluid.

Citation Format: Hironari Akasaka, Honami Naora. Impact of peritoneal lavage on omentum immune cell dynamics and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2521.
Abstract 2538: Role of Argonaute 2 in regulation of immune microenvironment in pancreatic cancer
Authors: Hon Jennifer , Kenum Carson , Bawa Pushpinder S. , Dommeti Vijaya L. , Sahu Anastasia A. , Gandham Miriam , Li Chi-Chiang , Taher Zainab I. , Zelenka-Wang Sylvia , Tien Jean C. , Shankar Sunita , Chugh Seema , Chinnaiyan Arul M.
Published: June 2022

KRAS mutations have been observed in nearly 95% of pancreatic ductal adenocarcinoma (PDAC), however targeting KRAS remains a major therapeutic challenge. Previous studies from our group have discovered a novel interaction between KRAS and Argonaute 2 (AGO2) and have uncovered a significant role of this interaction in regulating KRAS signaling. Knockout of AGO2 in genetically engineered mouse models of KRAS driven pancreatic and lung cancer dramatically impacted tumor progression. Intriguingly, in pancreatic cancer loss of AGO2 expression resulted in early pancreatic intraepithelial lesions (PanINs) that failed to progress to PDAC. We observed increased senescence in these AGO2 knockout lesions that abrogated PDAC progression. Further, there was pronounced infiltration of immune cells in pancreata lacking AGO2 in comparison to wild type. We particularly observed a 20-fold increase in natural killer (NK) cells population in pancreata lacking AGO2. This instigated us to evaluate potential role of AGO2 in regulating immune microenvironment in pancreatic cancer. Gene set enrichment analysis of AGO2 knockout SW1990 PDAC cells revealed significant upregulation of inflammatory and interferon response pathways. We observed increased expression of several signaling proteins implicated in immune activation pathways. Further characterization of these knockout cells indicated upregulation of MHC proteins that consequently added to mechanistic insights. Additionally, we are also exploring syngeneic models of pancreatic cancer and we will present findings from our ongoing studies to evaluate the impact of AGO2 knockout on immune axis. Overall, our findings suggest potential involvement of AGO2 in regulating immune microenvironment in pancreatic cancer.

Citation Format: Jennifer Hon, Carson Kenum, Pushpinder S. Bawa, Vijaya L. Dommeti, Anastasia A. Sahu, Miriam Gandham, Chi-Chiang Li, Zainab I. Taher, Sylvia Zelenka-Wang, Jean C. Tien, Sunita Shankar, Seema Chugh, Arul M. Chinnaiyan. Role of Argonaute 2 in regulation of immune microenvironment in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2538.
Abstract 2551: Sexual dimorphic effects of tumour microenvironment dictate melanoma progression and therapy resistance
Authors: Chhabra Yash , Fane Mitchell , Huser Laura , Zabransky Daniel , Brezka Megan , Marino Gloria , Carey Alexis , Weeraratna Ashani
Published: June 2022

Melanoma is a disease of ageing with elderly patients showing poorer prognosis than younger patients. The role of the ageing microenvironment in melanoma progression is now recognized where aged stromal cells undergo cellular and molecular changes that are tumour permissive. However, sex-based differences in melanoma incidence and outcome have also been observed even when adjusted for several prognostic factors including age, with men having poorer outcomes compared to women. While the mechanisms underlying this disparity are not well understood but attributed to sex-specific behaviors as well as to the biologically intrinsic differences. In addition, most preclinical studies have ignored the contribution of the host sex and age to therapy response, with majority of the studies typically performed in 6-8 week old male mice. Dermal fibroblasts (dFs) are established to have profound impact on melanoma progression. We examined if both sex-dependent and age-related changes in dFs can alter the course of melanoma tumor growth, visceral metastasis, dormancy and variable responses to targeted therapy. We find that age-matched female dFs undergo early replicative senescence accompanied by reduced proliferation and morphology change. Despite being senescent, age-matched female dFs showed reduced SASP characteristics. This was assessed by testing dFs derived conditioned media on melanoma cells in spheroid invasion and resistance to BRAF/MEK inhibition which showed elevated expression of invasive drivers and oxidative stress from aged male dFs derived conditioned media. This was also apparent in vivo in syngeneic immunocompetent C57Bl/6 mice that showed reduced tumor growth and increased invasion specific to the aged male host. Our data provides an integrated view of how age and sex attributes of dFs within the microenvironment contribute to disease progression. Bridging this knowledge gap will improve patient stratification and assist in tailoring the therapy and achieve gender-equitable healthcare.

Citation Format: Yash Chhabra, Mitchell Fane, Laura Huser, Daniel Zabransky, Megan Brezka, Gloria Marino, Alexis Carey, Ashani Weeraratna. Sexual dimorphic effects of tumour microenvironment dictate melanoma progression and therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2551.
Abstract 2567: Targeting TBL1/β-catenin complex using peptides designed to competitively inhibit aberrant Wnt signaling in cancer
Authors: Rheinschmidt Shelby , Thode Trason , Sampson Samuel , Weston Alexis , Halder Tithi Ghosh , Ng Serina , Rodriguez del Villar Ryan , Kaadige Mohan , Zernov Anton , Machluf Marcelle , Soldi Raffaella , Sharma Sunil
Published: June 2022

Purpose: Canonical Wnt signaling is a key cascade in regulating development and stemness, however it can become dysregulated and tightly associated with oncogenesis. In human cancers, Wnt/β-catenin signaling is highly activated due to mutations of members associated with the pathway such as adenomatous polyposis coli (APC) and β-catenin. Transducin β-like protein 1 (TBL1) and highly related TBLR1, form a complex with β-catenin facilitating the translocation of β-catenin to the nucleus. The formation of this complex protects β-catenin from degradation by SIAH-1 ubiquitinase and aids β-catenin binding to TCF/LEF transcription factors to transcribe downstream Wnt target genes. We recently reported that the destruction of the TBL1/β-catenin complex using a small molecule results in ubiquitination of β-catenin and inhibition of downstream signaling. As an alternative approach, we designed a series of novel peptides to interfere with binding of β-catenin with TBL1.

Methods: Homologous competitive ELISA and thermal shift assays were performed to identify potential peptides that were able to displace β-catenin from TBL1. Additionally, we evaluated the ability of the peptides to disrupt the TBL1/β-catenin complex by performing pull-down assays and monitored the levels of β-catenin ubiquitination in response to peptide treatment in colon cell lines. To assess the levels of TCF/LEF transcriptional activation downstream the Wnt/β-catenin pathway, we performed TOPFlash assays in Wnt-activated cells transfected with plasmids for peptide expression.

Results: Our preliminary results identified a set of peptides that efficiently displaces β-catenin from TBL1. Homologous competitive ELISA showed that the peptides were able to displace β-catenin from TBL1 in a dose dependent manner. The top five peptide hits were then tested for their ability to inhibit Wnt signaling using the TOPFlash assay. Among these, one peptide in particular showed high efficiency in inhibiting TCF/LEF transcriptional activation downstream the Wnt/β-catenin pathway. Our study suggests that β-catenin therapeutic peptides may represent a new and exciting approach for Wnt driven cancer therapy.

Citation Format: Shelby Rheinschmidt, Trason Thode, Samuel Sampson, Alexis Weston, Tithi Ghosh Halder, Serina Ng, Ryan Rodriguez del Villar, Mohan Kaadige, Anton Zernov, Marcelle Machluf, Raffaella Soldi, Sunil Sharma. Targeting TBL1/β-catenin complex using peptides designed to competitively inhibit aberrant Wnt signaling in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2567.
Abstract 2583: Utilization of a novel double knock-in humanized mouse model to evaluate the efficacy of ipilimumab and pembrolizumab against EMT-6 mammary carcinoma tumors
Authors: Stackhouse Murray , Kuta Mariah , Kolb Joseph , Scatigno Sydney , Blackwell Amber , Hall Chassidy , Dowdy Christopher , Sil Payel , Gietl Diana , Festin Steve , De Marval Paula L. Miliani
Published: June 2022

The cancer immunotherapy field has seen an increasing demand for identifying and characterizing pre-clinical models to evaluate the efficacy of the checkpoint inhibitors such as, ipilimumab and pembrolizumab. PBMC- or CD34-humanized mouse models are invaluable tools to assess effector functions of human T-cells to immune check point inhibitors (ICI). However, these models offer limited understanding of the overall potential therapeutic activity as they lack a complete repertoire of human immune cells required to elicit a full immune response. Thus, the development of multigenic humanized knock-in models provide a unique approach to evaluate the efficacy of ICI specific for human cancer targets in a fully immunocompetent organism. Here we describe the response of the EMT-6 murine breast cancer tumor model to ipilimumab and pembrolizumab in a novel hPDCD1/hCTLA4-KI double knock-in humanized BALB/c mouse model. EMT-6 tumors were implanted subcutaneously in the flank of hPDCD1/hCTLA4-KI mice. Three days post-implant we initiated treatment with ipilimumab or pembrolizumab alone and in combination. We observed significant ipilimumab anti-tumor activity, whereas pembrolizumab did not show efficacy despite effective T-cell PD-1 blockade. Unexpectedly, sudden respiratory distress occurred in animals receiving the 4th dose of combination therapy and the treatment was discontinued. To assess the immune phenotypic profiles in response to ipilimumab and/or pembrolizumab therapies, we performed multi-parameter flow cytometry analysis of peripheral blood, spleens and tumors. Additionally, we performed histopathological evaluation of lung tissues to assess potential neutrophil infiltration-mediated anaphylaxis in the mice treated with the combination treatment. The preliminary results highlight the value of the hPDCD1/hCTLA4-KI double knock-in humanized Balb/c model as a tool to evaluate human-specific immune-checkpoint based modalities. These models represent some of the difficulties faced in treating refractory tumors as well as reproducing some of the adverse effects seen in the clinic.

Citation Format: Murray Stackhouse, Mariah Kuta, Joseph Kolb, Sydney Scatigno, Amber Blackwell, Chassidy Hall, Christopher Dowdy, Payel Sil, Diana Gietl, Steve Festin, Paula L. Miliani De Marval. Utilization of a novel double knock-in humanized mouse model to evaluate the efficacy of ipilimumab and pembrolizumab against EMT-6 mammary carcinoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2583.