Metastasis In Response Research Articles

Abstract 2680: Combination of the Rac/Cdc42 inhibitor MBQ-167 with paclitaxel reduces metastasis in triple negative breast cancer

Abstract Metastasis represents a therapeutic challenge, especially in the case of triple negative breast cancer (TNBC), where patients often show recurrence and metastasis due to resistance to chemotherapy. The frontline therapy in TNBC is Paclitaxel, which targets actively dividing cells inducing subsequent apoptosis. Since dormant cells or metastatic cancer cells are not targeted by Paclitaxel, this strategy selects for non-dividing stem cells or more motile metastatic cells. Therefore, targeted treatment options are needed to overcome the recurrence and spreading of metastatic cells. We recently reported on MBQ-167, a Rac/Cdc42 inhibitor, which is highly effective in reducing tumor growth and metastasis in mouse models of TNBC (Cruz-Collazo et al., 2021, Molecular Cancer Therapeutics). Unpublished data show that in the MDA-MB-231 human TNBC model, MBQ-167 is as effective as Paclitaxel in reducing tumor growth and is more effective in metastasis prevention. Therefore, we hypothesize that MBQ-167 is a viable candidate for combined therapy with Paclitaxel and will reduce metastasis in TNBC. The purpose of this study is to test MBQ-167 in combination with Paclitaxel in the EGFR overexpressing MDA-MB-468 human TNBC cell line, which is highly malignant and also expresses the Rac1.B oncogenic splice variant. We found that MBQ-167 inhibits Rac, Rac1.B, and Cdc42 activation in this cell line. Next, we tested individual or combined MBQ-167 (50 mg/kg 5X a week) via oral administration and Paclitaxel (10 mg/kg 1X a week) via intraperitoneal administration in SCID mice bearing luciferase-tagged MDA-MB-468 tumors. Tumor growth and metastases were analyzed by chemiluminescence imaging followed by H&E staining of extracted lungs at necropsy. Similar to our studies with the syngeneic 4T-1/Balb/C model, in this MDA-MB-468 model, Paclitaxel increased metastasis while single agent MBQ-167 significantly reduced metastasis. MBQ-167 in combination with Paclitaxel reduced the enhanced lung metastasis in response to Paclitaxel. In conclusion, this study validates the clinical testing of MBQ-167 in combination with Paclitaxel as a potential combination therapy for TNBC. Citation Format: Ailed M. Cruz-Collazo, Nilmary Grafals, Luis D. Borrero, Suranganie Dharmawardhane. Combination of the Rac/Cdc42 inhibitor MBQ-167 with paclitaxel reduces metastasis in triple negative breast 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 2680.

Disrupting metformin adaptation of liver cancer cells by targeting the TOMM34/ATP5B axis

Metformin, a well‐known antidiabetic drug, has been repurposed for cancer treatment; however, recently observed drug resistance and tumor metastasis have questioned its further application. Here, we found that long‐term metformin exposure led to metabolic adaptation of hepatocellular carcinoma (HCC) cells, which was characterized by an obvious epithelial–mesenchymal transition (EMT) phenotype and compensatory elevation of oxidative phosphorylation (OXPHOS). TOMM34, a translocase of the outer mitochondrial membrane, was upregulated to promote tumor metastasis in response to metformin‐induced metabolic stress. Mechanistically, TOMM34 interacted with ATP5B to preserve F1FO‐ATPase activity, which conferred mitochondrial OXPHOS and ATP production. This metabolic preference for OXPHOS suggested a large requirement of energy supply by cancer cells to survive and spread in response to therapeutic stress. Notably, disturbing the interaction between TOMM34 and ATP5B using Gboxin, a specific OXPHOS inhibitor, increased sensitivity to metformin and suppressed tumor progression both in vitro and in vivo. Overall, this study demonstrates a molecular link of the TOMM34/ATP5B‐ATP synthesis axis during metformin adaptation and provides promising therapeutic targets for metformin sensitization in cancer treatment.

Abstract 4065: Efficacy of the Rac/Cdc42 inhibitor MBQ-167 in combination therapy with Paclitaxel

Abstract Triple negative breast cancer (TNBC) represents a challenge because of its aggressiveness and resistance to standard chemotherapy. Since TNBC lacks targeted therapy options, the frontline therapy is Paclitaxel, which targets actively dividing cells inducing subsequent apoptosis. Since dormant cells or metastatic cancer cells are not targeted by Paclitaxel, more targeted treatment options are needed. MBQ-167, a small molecule developed by us to target the metastasis drivers Rac and Cdc42, is highly effective in reducing tumor growth and metastasis in mouse models of TNBC (Cruz-Collazo et al., 2021, Molecular Cancer Therapeutics). The purpose of this study is to test the hypothesis that MBQ-167 is a viable candidate for combined therapy with Paclitaxel in TNBC. Therefore, we tested the efficacy of MBQ-167 in combination with Paclitaxel in MDA-MB-231 human TNBC cells via MTT viability assays and caspase 3/7 apoptosis, using effective concentrations of MBQ-167 and/or Paclitaxel for 24, 48, 96 & 120 hrs. We found that individual MBQ-167 or the combination therapy was more effective at 48-120 hrs at reducing cell viability by ~80% compared to ~60% by Paclitaxel treatment alone and significatively increasing apoptosis. Next, we tested individual or combined MBQ-167 (5 mg/kg 5X a week) and Paclitaxel (10 mg/kg 1X a week) on SCID mice bearing MDA-MB-231 tumors, via intraperitoneal administration. We report a significant reduction in tumor growth and lung metastasis in response to combined MBQ-167 and Paclitaxel compared to either treatment alone. A more aggressive syngeneic model of 4T-1 mouse breast cancer cells in BALB/c immunocompetent model was used to determine the direct effect of MBQ-167 and Paclitaxel on metastasis. When 4T-1 tumors were surgically removed and MBQ-167, Paclitaxel, or the combination administered for 17 days, the combination treatment, but not individual compounds, significantly reduced lung metastases by ~80%. In conclusion, this study validates the clinical testing of MBQ-167 in combination with Paclitaxel as a potential therapeutic for TNBC. Citation Format: Ailed M. Cruz-Collazo, Olga Katsara, Elizabeth Salvo, Jean Ruiz, Robert Schneider, Suranganie Dharmawardhane. Efficacy of the Rac/Cdc42 inhibitor MBQ-167 in combination therapy with Paclitaxel [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 4065.

Microenvironmental Landscape of Human Melanoma Brain Metastases in Response to Immune Checkpoint Inhibition.

Melanoma-derived brain metastases (MBM) represent an unmet clinical need because central nervous system progression is frequently an end stage of the disease. Immune checkpoint inhibitors (ICI) provide a clinical opportunity against MBM; however, the MBM tumor microenvironment (TME) has not been fully elucidated in the context of ICI. To dissect unique elements of the MBM TME and correlates of MBM response to ICI, we collected 32 fresh MBM and performed single-cell RNA sequencing of the MBM TME and T-cell receptor clonotyping on T cells from MBM and matched blood and extracranial lesions. We observed myeloid phenotypic heterogeneity in the MBM TME, most notably multiple distinct neutrophil states, including an IL8-expressing population that correlated with malignant cell epithelial-to-mesenchymal transition. In addition, we observed significant relationships between intracranial T-cell phenotypes and the distribution of T-cell clonotypes intracranially and peripherally. We found that the phenotype, clonotype, and overall number of MBM-infiltrating T cells were associated with response to ICI, suggesting that ICI-responsive MBMs interact with peripheral blood in a manner similar to extracranial lesions. These data identify unique features of the MBM TME that may represent potential targets to improve clinical outcomes for patients with MBM.

Osteocytic Connexin Hemichannels Modulate Oxidative Bone Microenvironment and Breast Cancer Growth.

Simple SummaryEstrogen deficiency with increased oxidative stress is associated with various pathological conditions, including cancers. Oxidative stress (OS) is elevated in the bone microenvironment with estrogen deficiency, but the impact of OS on tumor growth in bone remains largely unknown. In our research, we found that elevated oxidative stress inhibits tumor cell growth and the lack of Cx43 hemichannel function in bone osteocytes could mediate this effect. Impairment of Cx43 hemichannels either under oxidative stress or in Cx43-hemichannel-impaired transgenic mice under estrogen deficiency led to the inhibition of breast cancer growth. Treatment with an antioxidant reverses the effect, showing an increase in tumor growth in mice. This study unveiled a new mechanism regarding the role of osteocytic Cx43 hemichannels in modulating an oxidative bone microenvironment and the response of breast cancer cells to local oxidative stress.Osteocytes, the most abundant bone cell types embedded in the mineral matrix, express connexin 43 (Cx43) hemichannels that play important roles in bone remodeling and osteocyte survival. Estrogen deficiency decreases osteocytic Cx43 hemichannel activity and causes a loss in osteocytes’ resistance to oxidative stress (OS). In this study, we showed that OS reduced the growth of both human (MDA-MB-231) and murine (Py8119) breast cancer cells. However, co-culturing these cells with osteocytes reduced the inhibitory effect of OS on breast cancer cells, and this effect was ablated by the inhibition of Cx43 hemichannels. Py8119 cells were intratibially implanted in the bone marrow of ovariectomized (OVX) mice to determine the role of osteocytic Cx43 hemichannels in breast cancer bone metastasis in response to OS. Two transgenic mice overexpressing dominant-negative Cx43 mutants, R76W and Δ130-136, were adopted for this study; the former inhibits gap junctions while the latter inhibits gap junctions and hemichannels. Under normal conditions, Δ130-136 mice had significantly more tumor growth in bone than that in WT and R76W mice. OVX increased tumor growth in R76W but had no significant effect on WT mice. In contrast, OVX reduced tumor growth in Δ130-136 mice. To confirm the role of OS, WT and Δ130-136 mice were administered the antioxidant N-acetyl cysteine (NAC). NAC increased tumor burden and growth in Δ130-136 mice but not in WT mice. Together, the data suggest that osteocytes and Cx43 hemichannels play pivotal roles in modulating the oxidative microenvironment and breast cancer growth in the bone.

LMD-03. Single cell analysis reveals how therapy remodels the tumor microenvironment in melanoma CNS metastases and uncovers a novel predictor of improved survival

We interrogated the microenvironment of 43 clinical samples from melanoma skin, brain (MBM) and leptomeningeal metastases (LMM) using single-cell RNA-seq analysis to determine how therapeutic intervention shaped the immune environment and affected patient survival. LMM is a poorly-characterized, devastating complication of late-stage disease, typically refractory to treatment and associated with dismal survival time. Analysis of serial specimens over the course of therapy demonstrated reductions in melanoma cells and macrophages, coupled with increased levels of T cells and dendritic cells in the CSF of a rare extraordinary responder, whereas typical poor survivors showed no improvement in T cell responses. In MBM patients, both targeted therapy and immunotherapy was associated with increased immune infiltrate. Treatment with targeted therapy was associated with an enrichment of CD8 T cells, while immunotherapy was associated with a more diverse lymphocyte landscape and higher numbers of antibody-producing cells. These findings were confirmed by multiplex-IF staining of patient specimens and using an immune-competent mouse model of MBM. Interestingly, a history of prior radiation therapy was associated with a diminished myeloid compartment. Although immune infiltrate was significantly lower in the brain compared to skin tumors, the phenotypic make-up of the lymphocyte compartment was quite similar, suggesting that the immune cells may have trafficked from the periphery to the brain post-therapy. Correlation analysis across the entire immune landscape identified the presence of a rare, novel population of dendritic cells (DC3s) to be correlated with increased overall survival, regardless of disease site/treatment. The presence of DC3s positively regulated the immune environment of both patient samples and preclinical melanoma models through modulation of activated T cells and MHC expression in the tumor. Overall, we present the first ever comprehensive single-cell atlas of the tumor microenvironment in melanoma CNS metastases in response to therapy.

Abstract 2405: Metabolic plasticity of ovarian metastases in response to adherence and physiologically relevant culture conditions

Abstract Ovarian cancer is typically diagnosed at late stages with a poor prognosis due to high recurrence. Exfoliation from the primary tumor results in aggregation of ovarian cancer cells into spheroids in the peritoneal cavity, which then adhere to secondary sites to form metastatic outgrowth. Spheroids in the peritoneal cavity are exposed to hypoxic, low glucose conditions. Sphingosine-1-phosphate (S1P), increased in the ascites fluid of ovarian cancer patients, has been shown to increase tumor cell survival and tumorigenesis through changes in mitochondrial function. However, little is known about these effects in ovarian cancer spheroids, particularly upon secondary adherence and outgrowth in physiological conditions. Changes in spheroid metabolism in response to treatment with S1P, hypoxia, low glucose, and obese peritoneal serous fluid (PSF) in both free and adherent states was investigated. Using a late-stage and tumor-initiating murine ovarian cancer model, spheroids were subjected to treatment with S1P or obese PSF as free or adherent spheroids. Using a Seahorse XFe96, we investigated changes in metabolism over time trials measured after 24 hour treatment for free spheroids and after different time points of adherence (4, 8, 12, and 24 hours). Data show changes in basal respiration, maximal oxygen consumption rate, and spare respiratory capacity of spheroids over adherence time when treated with S1P and PSF. This suggests that the cells respond to energetic demand with metabolic flexibility as the spheroids adhere to new surfaces. The identification of the regulation of these events may help elucidate the flexibility that takes place during metastasis in culture that more closely mimics physiological conditions, providing a foundation for future interventions for preventative therapies for ovarian cancer with dietary sphingolipids and possible treatment targets. Citation Format: Stephanie Edwards Compton, Joseph Grieco, Eva M. Schmelz. Metabolic plasticity of ovarian metastases in response to adherence and physiologically relevant culture conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2405.

The Effects of Localized Heat on the Hallmarks of Cancer

AbstractHeat has been used to treat tumors for thousands of years. There are reports of the Egyptians and Greek philosophers using such treatments as far back as 3000 BC and 500 BC respectively for various solid tumors. Albeit, in these cases, the treatment was not very controlled and consisted of hot sticks or blades placed against tissue in order to thermally ablate the tumor. It was not until recent times that the application of heat through various mediums enabled a more controlled, localized, and consistent method of treating tumors. While the therapeutic potential of this treatment has become more apparent, the mechanisms related to its efficacy are only recently beginning to surface. This review discusses the evidence associated with the effects of localized heat on the hallmarks of cancer. Key literature describing modulations to vasculature, cell viability, DNA damage and repair, metabolism, immune system, and tumor metastasis in response to heat will be reviewed along with considerations for its optimal implementation in the clinic to enhance the efficacy of conventional treatments.

Isoliquiritigenin Reverses Epithelial-Mesenchymal Transition Through Modulation of the TGF-β/Smad Signaling Pathway in Endometrial Cancer.

Simple SummaryThe high recurrence risk and poor prognosis of metastatic endometrial cancer are the main focus of interventional therapy. In view of this, we established in vitro and in vivo metastasis models and explored the underlying mechanisms of the epithelial-mesenchymal transition (EMT) process, cell migration ability, and metastasis in response to isoliquiritigenin (ISL). The presented in vitro and in vivo preclinical studies both demonstrated that ISL efficiently suppressed endometrial cancer cell migration and reduced the HEC-1A-LUC tumor metastasis in nude mice through inhibiting TGF-β/Smad signaling pathway. These findings shed the light for further research to highlight the ISL potential in endometrial cancer metastasis.Endometrial cancer is a common gynecological cancer with a poor prognosis, mostly attributed to tumor metastasis. Epithelial–mesenchymal transition (EMT) can be mediated via transforming growth factor beta (TGF-β) signaling pathway, facilitating the ability of cancer cell invasion and migration. Isoliquiritigenin (ISL) is a flavonoid derived from licorice with reported antineoplastic activities. This study aims to investigate the anti-metastatic potential of ISL on endometrial cancer both in vitro and in vivo. First, human endometrial cancer cell lines (HEC-1A, Ishikawa, and RL95-2) were treated with ISL and then subjected to functional assays such as migration assay as well as molecular analyses including immunoblotting, immunofluorescence and RT-qPCR. In addition, HEC-1A-LUC cells were implanted into female nude mice and treated with ISL by intraperitoneal injection for four weeks. Results showed that ISL inhibited cell migration and reversed the effect of TGF-β on the expression of E-cadherin, N-cadherin, vimentin, α-SMA, p-Smad3, and TWIST1/2 In vitro. Interestingly, In vivo study revealed that ISL reduced peritoneal dissemination and serum level of TGF-β1, as well as decreased the expression levels of N-cadherin, p-Smad2/3, TWIST1/2, while increased E-cadherin. Overall, ISL reverses the EMT through targeting the TGF-β/Smad signaling pathway and features a potential therapeutic treatment for metastatic endometrial cancer.

Abstract SP159: EMT

Abstract During metastasis, epithelial tumor cells dissociate from each other, disseminate into the systemic circulation, and then establish secondary tumors in distant sites. A developmental program termed Epithelial-Mesenchymal Transition (EMT) is implicated in promoting the dissemination of single carcinoma cells during metastasis. Our recent studies aim to understand how EMT is dynamically regulated in response to signals from the tumor microenvironment and from the intracellular machineries to impact EMT and tumor metastasis. Specifically, I will present a mechanotransduction pathway that senses and transmits mechanincal cues from stiff matrix in the tumor microenvironment to promote EMT and invasion during tumor progression. Breast tumors are often detected due to their apparent “hardness” compared to normal tissues, and increasing matrix stiffness correlates with distant metastasis and poor survival in breast cancer patients. These observations raise the question of how mechanical forces generated by the rigid tumor ECM impact tumor progression and metastasis. In a hydrogel coupled with 3D Matrigel overlay culture system that recapitulates the range of physiological stiffness from normal mammary glands to breast tumors, increased ECM stiffness triggers Epithelial-Mesenchymal Transition (EMT), invasion and metastasis. We previously reported that the transcription factor Twist1 is an essential mechano-mediator that promotes EMT and metastasis in response to ECM stiffness. High matrix stiffness promotes Twist1 nuclear localization through phosphorylation-dependent regulation of its interaction with cytoplasmic anchor G3BP2. Using kinase screening and protein affinity purification, we identified a novel mechanoresponsive protein complex including an ephrin receptor and a Src-family kinase that promotes Twist1 phosphorylation and subsequent nuclear translocation in response to high matrix rigidity. Pharmacological and genetic inhibition of this mechanotransduction pathway blocks ECM stiffness-induced EMT and invasion in vitro and inhibits metastasis in human breast tumor xenografts. Our findings reveal a novel mechanotransduction pathway that responds to mechanical signals from the tumor microenvironment to drive EMT, invasion, and metastasis. Citation Format: J Yang. EMT [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP159.

Tall-cell variant of papillary thyroid carcinoma: literature review and case reports

The tall-cell variant of papillary thyroid carcinoma is a rare malignancy characterized by an aggressive course, low relapse-free survival, early metastasis, and frequent development of radioiodine refractoriness. We report two cases of tall-cell papillary thyroid carcinoma in patients who had disease progression after combination treatment and started to receive lenvatinib. We observed partial and complete regression of metastases in response to lenvatinib. Our findings are consistent with the results of other studies assessing treatment efficacy for disseminated tall-cell papillary thyroid carcinoma.

NIMG-04. MRI EVALUATION OF THE EFFECT OF WHOLE BRAIN RADIOTHERAPY ON BRAIN METASTASIS

Abstract Poor prognosis of breast cancer brain metastasis partially results from the inability of therapeutics to penetrate the Blood-Brain-Barrier (BBB). Whole Brain Radiotherapy (WBRT) is standard-of-care for brain metastasis, however the early effects of fractionated WBRT on brain metastases have not been evaluated. Here, we applied longitudinal MRI to study WBRT’s effect on tumor volume, BBB permeability, and tumor-cell killing using anatomic, Dynamic Contrast Enhanced (DCE), and Diffusion-weighted (DW) MRI, evaluating tumor volume, permeability, and cytotoxic effect, respectively. The BCBM mouse model was established with left ventricle MDA-MB231-Br cell injection. MRI occurred before and 24h after 3 daily fractionated 4Gy WBRT (2.4Gy/min) or sham irradiation. In addition to anatomic MRI, DW MRI measured tissue ADC to study cytotoxic effect, and DCE MRI measured BBB permeability, quantified as the permeability variable Ktrans. Before treatment, T2-weighted images revealed hyperintense multifocal lesions in brains, and many had intact BBB indicated by no T1-w contrast-enhancement. WBRT tumors were smaller than sham irradiated tumors after 3 days (p< 0.05), while no difference in the number of new lesions was observed. DW MRI demonstrated increased ADC in WBRT tumors (p< 0.05), correlating well with elevated Caspase-3 staining of apoptotic cells. T1-w contrast enhanced images of tumors after treatment revealed no differences in the population of contrast-enhanced tumors between the WBRT and sham radiation groups. However, quantitative DCE MRI showed higher Ktrans in WBRT treated tumors relative to sham treated lesions, and an increase in Ktrans of WBRT tumors (p< 0.05), despite high permeability heterogeneity both inter- and intra-tumorally, indicating remaining impermeable BBB. In addition to monitoring tumor volume, longitudinal MRI provided non-invasive assessments of temporal and spatial changes in cellularity and vascular permeability of brain metastasis in response to WBRT, which may prove to be useful for defining an ideal treatment window for potential combination BCBM treatments.

Control and Toxicity in Melanoma Versus Other Brain Metastases in Response to Combined Radiosurgery and PD-(L)1 Immune Checkpoint Inhibition

PurposePrior studies have mixed conclusions about the efficacy and central nervous system (CNS) toxicity profile of combining radiosurgery with anti-programed cell death 1 (PD-1) immune checkpoint inhibition (ICI) for brain metastases. This study evaluates the safety and efficacy of combined radiosurgery and anti-PD-1 ICI for melanoma, non-small cell lung cancer (NSCLC), and renal cell carcinoma (RCC) brain metastases (BM). Methods and MaterialsForty-one patients with 153 radiation naïve melanoma BM and 33 patients with 118 BM of NSCLC and RCC origin from 2014 through 2019 received radiosurgery and either anti PD-1 receptor inhibition or anti PD-L1 inhibition targeting the PD-1 ligand with less than 4 months separating either therapy. Similar to Radiation Therapy Oncology Group 9005, high-grade CNS toxicity was defined as irreversible grade 3 or any grade 4/5 neurologic event. Salvage resection revealing necrosis and viable tumor was considered grade 4 toxicity and local failure. An increase in greatest cross-sectional diameter of 25% on contrasted magnetic resonance imaging was designated as a local failure. ResultsMedian follow-up was 10 months (range, 1-41 months). Local control was estimated to be 90.3% at 1 year. Distant control was 38.8% at 1 year, and neither local nor distant control were significantly influenced by limiting steroids to the day of treatment (P = .55, .52 respectively). One-year freedom from high-grade toxicity was 90.4% for patients and 94.6% for tumors. Though melanoma accounted for 41 (55%) patients and 153 (56%) tumors, it accounted for all high-grade toxicities (P = .03). These patients had some combination of high tumor burden, aggressive steroid taper, and treatment with ipilimumab. ConclusionsStereotactic radiosurgery combined with anti-PD-1 ICI appears to result in a high rate of local tumor control and a low rate of high-grade CNS toxicity, comparable to historical series with radiosurgery alone. High-grade toxicity is more likely in melanoma than RCC and NSCLC. Coming prospective studies will shed light on further questions about treatment timing, steroids, and response.

Abstract 2738: Role of KLF8 for drug-resistant metastasis of breast cancer

Abstract Metastasis and drug resistance are intertwined processes that are responsible for the vast majority of patient deaths from breast cancer. The underlying mechanisms still need to be investigated extensively. We previously demonstrated that KLF8 activates CXCR4 transcription in metastatic breast cancer. Here, we report a novel signaling mechanism that turns a localized metastasis into multiple organ metastases in response to chemotherapy. We show that KLF8 expression in breast cancer cells can be over-induced by chemotherapeutic drugs. Data from large-cohorts of patients indicates that post-chemotherapy there is a close correlation between the aberrant high levels of KLF8 and CXCR4, and this correlation is well associated with drug resistance, metastasis, and poor prognosis. To mimic this upregulation, we overexpressed KLF8 or CXCR4 in a human breast cancer cell line known to metastasize to the lungs only after i.v. injection in the nude mice. Surprisingly, we found that these KLF8 or CXCR4 overexpressing cells metastasized all over the body after orthotopically implanted. Tube formation assay, Ki67 staining, and western blotting revealed that KLF8 or CXCR4 overexpression promoted angiogenic vascular growth involving increased expression and secretion of VEGF protein. We also performed the matrigel-on-top 3-D culture to mimic the colonization step of metastasis and found that KLF8 or CXCR4 overexpression strongly enhances the formation of filopodium-like protrusions and proliferation via CXCR4 stimulation. Taken together, these results suggest that the chemo-induction of KLF8 upregulation is critical for drug resistance and systemic metastasis through enhanced tumor angiogenesis and colonization via CXCR4 over-activation. This discovery identifies the KLF8-CXCR4 signaling axis as a potentially new therapeutic target for breast cancer. Citation Format: Jie Hao, Heng Lu, Debarati Mukherjee, Aiqin Sun, Lin Yu, Jihe Zhao. Role of KLF8 for drug-resistant metastasis of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2738.

CSC Radioresistance: A Therapeutic Challenge to Improve Radiotherapy Effectiveness in Cancer.

Radiotherapy (RT) is a modality of oncologic treatment that can be used to treat approximately 50% of all cancer patients either alone or in combination with other treatment modalities such as surgery, chemotherapy, immunotherapy, and therapeutic targeting. Despite the technological advances in RT, which allow a more precise delivery of radiation while progressively minimizing the impact on normal tissues, issues like radioresistance and tumor recurrence remain important challenges. Tumor heterogeneity is responsible for the variation in the radiation response of the different tumor subpopulations. A main factor related to radioresistance is the presence of cancer stem cells (CSC) inside tumors, which are responsible for metastases, relapses, RT failure, and a poor prognosis in cancer patients. The plasticity of CSCs, a process highly dependent on the epithelial–mesenchymal transition (EMT) and associated to cell dedifferentiation, complicates the identification and eradication of CSCs and it might be involved in disease relapse and progression after irradiation. The tumor microenvironment and the interactions of CSCs with their niches also play an important role in the response to RT. This review provides a deep insight into the characteristics and radioresistance mechanisms of CSCs and into the role of CSCs and tumor microenvironment in both the primary tumor and metastasis in response to radiation, and the radiobiological principles related to the CSC response to RT. Finally, we summarize the major advances and clinical trials on the development of CSC-based therapies combined with RT to overcome radioresistance. A better understanding of the potential therapeutic targets for CSC radiosensitization will provide safer and more efficient combination strategies, which in turn will improve the live expectancy and curability of cancer patients.

Matrix Rigidity Controls Epithelial-Mesenchymal Plasticity and Tumor Metastasis via a Mechanoresponsive EPHA2/LYN Complex.

Mechanical cues from the extracellular matrix (ECM) regulate various cellular processes via distinct mechanotransduction pathways. In breast cancer, increased ECM stiffness promotes epithelial-to-mesenchymal transition (EMT), cell invasion, and metastasis. Here, we identify a mechanosensitive EPHA2/LYN protein complex regulating EMT and metastasis in response to increasing ECM stiffness during tumor progression. High ECM stiffness leads to ligand-independent phosphorylation of ephrin receptor EPHA2, which recruits and activates the LYN kinase. LYN phosphorylates the EMT transcription factor TWIST1 to release TWIST1 from its cytoplasmic anchor G3BP2 to enter the nucleus, thus triggering EMT and invasion. Genetic and pharmacological inhibition of this pathway prevents breast tumor invasion and metastasis invivo. In human breast cancer samples, activation of this pathway correlates with collagen fiber alignment, a marker of increasing ECM stiffness. Our findings reveal an EPHA2/LYN/TWIST1 mechanotransduction pathway that responds to mechanical signals from the tumor microenvironment to drive EMT, invasion, and metastasis.

Differential Change in Size of Human Papillomavirus-Positive Cystic Versus Solid Squamous Cell Carcinoma Lymph Node Metastases in Response to Induction Chemotherapy.

According to certain cancer treatment protocols, the response to induction chemotherapy of lymph node metastases based on radiographic measurements guides further management. The aim of this study is to verify the observation that cystic metastatic lymph nodes tend not to shrink as rapidly as solid metastatic lymph nodes in response to induction chemotherapy in patients with human papillomavirus-related oropharyngeal squamous cell carcinoma. The lymphadenopathy in a cohort of patients from a clinical trial with human papillomavirus-related oropharyngeal squamous cell carcinoma with both baseline and postinduction chemotherapy (carboplatin/paclitaxel/cetuximab) contrast-enhanced neck computed tomography was retrospectively reviewed. The appearance of the metastatic lymph nodes on computed tomography was characterized as cystic or solid. A cystic lymph node was defined as having a hypoattenuating component greater than 20% of the total volume. The rates of short-axis and volume changes of cystic and solid lymph nodes were compared using 1-tailed t test. A total of 46 patients were included in this study, comprising 39 solid and 45 cystic lymph nodes. The rate of short-axis decrease was significantly greater for solid (1.33% per day) than cystic (1.08% per day) lymph nodes (P = 0.036). Likewise, the rate of volume decrease was significantly greater for solid (2.13% per day) than cystic (1.87% per day) lymph nodes (P = 0.014). This study suggests that in patients with human papillomavirus-related oropharyngeal squamous cell carcinoma solid lymph node metastases generally decrease in size at a greater rate than cystic lymph nodes after induction chemotherapy.

Abstract P2-20-03: Estrogen-depletion therapies prevent and delay progression of brain metastasis

Abstract Introduction: Triple negative breast cancers (TNBC, which lack estrogen receptor (ER), progesterone receptor and HER2), occur three times more frequently in premenopausal women and metastasize expeditiously to brain and lungs. Clinical and experimental evidence shows that estrogen, the main premenopausal ovarian hormone, promotes metastases of TNBC, through their action on the tumor microenvironment. We have shown that 17-β-Estradiol (E2) promotes experimental brain metastases (BM) of TNBC cells by inducing estrogen-receptor positive (ER+) astrocytes in the brain niche to secrete growth factors that promote cancer cell migration, invasion and growth. Given that 53% of pre-menopausal women with TNBC develops BM and 80% die within a year, it is now critical to define whether blocking estrogen signaling could have therapeutic value to prevent or delay progression of BM. Results: To assess whether E2-depletion could prevent brain metastatic colonization, brain trophic sublines of human TNBC (231BR), murine TNBC (4T1BR5), a non-selected TN cell line syngeneic to C57BL mice (E0771-GFP-luc) were intracardially (ic) injected two-days post-endocrine initiation, in ovariectomized (OVX) female mice treated with a) E2 (levels equivalent to those found in pre-menopausal women), b) placebo (OVX), or c) the aromatase-inhibitor (AI) letrozole (OVX + Letrozole) to block brain and peripheral E2-synthesis. BM were quantified histologically, via MRI or ex-vivo imaging of brains at euthanasia. E2-treated mice injected with E0771-GFP-luc cells showed an average of 6.6± 3.5 metastatic clusters per mice, compared to 3.6 ± 3.2 and 1.8 ± 1.8 in OVX mice alone or OVX + letrozole treated mice (a 3.6 fold increase, P=0.0013, E2 vs OVX + Let). Imaging of excised brains showed comparable results, with a mean total photon flux of 5.1 × 106 ± 5.3 × 106 in E2 vs 1.1 × 106 ± 1.5 × 106 in OVX + letrozole (P=0.0056). Similar preventive effects of E2-depletion were found in 4T1BR5 and 231BR-cell injected mice, using histological quantification and MRI as endpoints. To assess whether E2-depletion therapies could decrease brain metastatic progression in a therapeutic model mimicking the clinical setting, E0771-GFP-luc cells were injected in OVX-mice that had been supplemented with E2 and metastasis allowed to develop for 6 days. Mice were then randomized to a) E2, b) E2-withdrawal (E2WD) or c) E2WD+ letrozole, and then all mice were irradiated (whole brain irradiation, single-dose, 15 Gy) to mimic current standard of care for BMs. All mice were euthanized 10 days later, when E2-treated mice showed signs of CNS impairment. Mice that received E2WD+letrozole showed the lowest BM burden compared to mice that remained in E2-treatments, as assessed by histological quantification (0.7 ± 1.2 vs 6.5 ± 5.3 metastatic clusters, respectively, P=0.0132) and ex-vivo brain imaging (mean total-photon flux 3.4 × 105 ± 4.1 × 105 in E2WD + let vs 3.1 × 106 ± 1.4 × 106 in E2, P=0.037). Brain metastasis in TNBC often occur in women who also present with systemic metastatic disease, and E0771-GFP-luc cells can colonize multiple organs following ic injection. Thus, non-brain metastatic burden (systemic metastasis) was measured by in vivo imaging before euthanasia in this cohort. Unlike brain metastasis, E2WD or E2WD+letrozole showed no statistically significant effect on systemic metastatic burden (mean total flux 1.41 × 106 ± 0.9 × 106 in E2, vs 0.54 × 106 ± 2.6 × 106 in E2WD vs 0.79 × 106 ± 1.0 × 106 in E2WD+let, P=0.416), supporting the notion that brain-specific rather than systemic mechanisms drive progression of brain metastasis in response to E2. Conclusions: These studies have important clinical implications as they provide pre-clinical evidence that ovarian suppression in combination with AIs can prevent and delay progression of established brain metastasis in younger women with TNBC. Citation Format: Diana M. Cittelly, Maria J. Contreras-Zarate, Ricaurte A. Marquez-Ortiz, Nicole Day, Bebhinn Nagle, Ryan Ormond, Virginia F. Borges, Peter Kabos. Estrogen-depletion therapies prevent and delay progression of brain metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-20-03.

C3a elicits unique migratory responses in immature low-density neutrophils.

Neutrophils represent the immune system's first line of defense and are rapidly recruited into inflamed tissue. In cancer associated inflammation, phenotypic heterogeneity has been ascribed to this cell type, whereby neutrophils can manifest anti- or pro-metastatic functions depending on the cellular/micro-environmental context. Here, we demonstrate that pro-metastatic immature low-density neutrophils (iLDNs) more efficiently accumulate in the livers of mice bearing metastatic lesions compared with anti-metastatic mature high-density neutrophils (HDNs). Transcriptomic analyses reveal enrichment of a migration signature in iLDNs relative to HDNs. We find that conditioned media derived from liver-metastatic breast cancer cells, but not lung-metastatic variants, specifically induces chemotaxis of iLDNs and not HDNs. Chemotactic responses are due to increased surface expression of C3aR in iLDNs relative to HDNs. In addition, we detect elevated secretion of cancer-cell derived C3a from liver-metastatic versus lung-metastatic breast cancer cells. Perturbation of C3a/C3aR signaling axis with either a small molecule inhibitor, SB290157, or reducing the levels of secreted C3a from liver-metastatic breast cancer cells by short hairpin RNAs, can abrogate the chemotactic response of iLDNs both in vitro and in vivo, respectively. Together, these data reveal novel mechanisms through which iLDNs prefentially accumulate in liver tissue harboring metastases in response to tumor-derived C3a secreted from the liver-aggressive 4T1 breast cancer cells.

A Bioinspired Nanoprobe with Multilevel Responsive T1 -Weighted MR Signal-Amplification Illuminates Ultrasmall Metastases.

Metastasis remains the major cause of death in cancer patients. Thus, there is a need to sensitively detect tumor metastasis, especially ultrasmall metastasis, for early diagnosis and precise treatment of cancer. Herein, an ultrasensitive T1 -weighted magnetic resonance imaging (MRI) contrast agent, UMFNP-CREKA is reported. By conjugating the ultrasmall manganese ferrite nanoparticles (UMFNPs) with a tumor-targeting penta-peptide CREKA (Cys-Arg-Glu-Lys-Ala), ultrasmall breast cancer metastases are accurately detected. With a behavior similar to neutrophils' immunosurveillance process for eliminating foreign pathogens, UMFNP-CREKA exhibits a chemotactic "targeting-activation" capacity. UMFNP-CREKA is recruited to the margin of tumor metastases by the binding of CREKA with fibrin-fibronectin complexes, which are abundant around tumors, and then release of manganese ions (Mn2+ ) to the metastasis in response to pathological parameters (mild acidity and elevated H2 O2 ). The localized release of Mn2+ and its interaction with proteins affects a marked amplification of T1 -weighted magnetic resonance (MR) signals. In vivo T1 -weighted MRI experiments reveal that UMFNP-CREKA can detect metastases at an unprecedented minimum detection limit of 0.39 mm, which has significantly extended the detection limit of previously reported MRI probe.