Prognostic value of cerebrospinal fluid tumor cell count in leptomeningeal disease from solid tumors.

PURPOSE: Treatment decisions for leptomeningeal disease (LMD) rely on patient risk stratification, since clinicians lack objective prognostic tools. The introduction of rare cell capture technology for identification of cerebrospinal fluid tumor cells (CSF-TCs), such as CNSide assay, improved the sensitivity of LMD diagnosis, but prognostic value is unknown. This study assesses the prognostic value of CSF-TC density in patients with LMD from solid tumors. METHODS: We conducted a retrospective cohort study of patients with newly diagnosed or previously treated LMD from a single institution who had CNSide assay testing for CSF-TCs from 2020 to 2023. Univariable and multivariable survival analyses were conducted with Cox proportional-hazards modeling. Maximally-selected rank statistics were used to determine an optimal cutpoint for CSF-TC density and survival. RESULTS: Of 31 patients, 29 had CSF-TCs detected on CNSide. Median (interquartile range [IQR]) CSF-TC density was 67.8 (4.7-639) TCs/mL. CSF cytology was positive in 16 of 29 patients with positive CNSide (CNSide diagnostic sensitivity = 93.5%, negative predictive value = 85.7%). Median (IQR) survival from time of CSF-TC detection was 176 (89-481) days. On univariable and multivariable analysis, CSF-TC density was significantly associated with survival. An optimal cutpoint for dichotomizing survival by CSF-TC density was 19.34 TCs/mL. The time-dependent sensitivity and specificity for survival using this stratification were 76% and 67% at 6 months and 65% and 67% at 1 year, respectively. CONCLUSIONS: CSF-TC density may carry prognostic value in patients with LMD from solid tumors. Integrating CSF-TC density into LMD patient risk-stratification may help guide treatment decisions.

Keeping a track on leptomeningeal disease in non-small cell lung cancer: A single-institution experience with CNSideTM.

Background: Leptomeningeal disease (LMD) is a devastating complication for patients with advanced cancer. Diagnosis and monitoring the response to therapy remains challenging due to limited sensitivity and specificity of standard-of-care (SOC) diagnostic modalities, including cerebrospinal fluid (CSF) cytology, MRI, and clinical evaluation. These hindrances contribute to the poor survival of LMD patients. CNSide is a CLIA-validated test that detects and characterizes CSF-derived tumor cells and cell-free (cf) DNA. We performed a retrospective analysis on the utility of CNSide to analyze CSF obtained from advanced non-small cell lung cancer (aNSCLC) patients with suspected LMD treated at the Huntsman Cancer Institute in Salt Lake City, UT. Methods: CNSide was used to evaluate CSF from 15 patients with aNSCLC. CSF tumor cell quantification was performed throughout treatment for 5 patients. CSF tumor cells and cfDNA were characterized for actionable mutations. Results: In LMD-positive patients, CNSide detected CSF tumor cells in 88% (22/25) samples versus 40% (10/25) for cytology (matched samples). CSF tumor cell numbers tracked response to therapy in 5 patients where CNSide was used to quantify tumor cells throughout treatment. In 75% (9/12) of the patients, genetic alterations were detected in CSF, with the majority representing gene mutations and amplifications with therapeutic potential. The median survival for LMD patients was 16.1 m (5.2-NR). Conclusions: We show that CNSide can supplement the management of LMD in conjunction with SOC methods for the diagnosis, monitoring response to therapy, and identifying actionable mutations unique to the CSF in patients with LMD.

Investigating the contribution of hyaluronan to the breast tumour microenvironment using multiparametric MRI and MR elastography.

Hyaluronan (HA) is a key component of the dense extracellular matrix in breast cancer, and its accumulation is associated with poor prognosis and metastasis. Pegvorhyaluronidase alfa (PEGPH20) enzymatically degrades HA and can enhance drug delivery and treatment response in preclinical tumour models. Clinical development of stromal-targeted therapies would be accelerated by imaging biomarkers that inform on therapeutic efficacy in vivo. Here, PEGPH20 response was assessed by multiparametric magnetic resonance imaging (MRI) in three orthotopic breast tumour models. Treatment of 4T1/HAS3 tumours, the model with the highest HA accumulation, reduced T1 and T2 relaxation times and the apparent diffusion coefficient (ADC), and increased the magnetisation transfer ratio, consistent with lower tissue water content and collapse of the extracellular space. The transverse relaxation rate R2 * increased, consistent with greater erythrocyte accessibility following vascular decompression. Treatment of MDA-MB-231 LM2-4 tumours reduced ADC and dramatically increased tumour viscoelasticity measured by MR elastography. Correlation matrix analyses of data from all models identified ADC as having the strongest correlation with HA accumulation, suggesting that ADC is the most sensitive imaging biomarker of tumour response to PEGPH20.

PEGylated recombinant human hyaluronidase (PEGPH20) pre-treatment improves intra-tumour distribution and efficacy of paclitaxel in preclinical models.

BACKGROUND: Scarce drug penetration in solid tumours is one of the possible causes of the limited efficacy of chemotherapy and is related to the altered tumour microenvironment. The abnormal tumour extracellular matrix (ECM) together with abnormal blood and lymphatic vessels, reactive stroma and inflammation all affect the uptake, distribution and efficacy of anticancer drugs. METHODS: We investigated the effect of PEGylated recombinant human hyaluronidase PH20 (PEGPH20) pre-treatment in degrading hyaluronan (hyaluronic acid; HA), one of the main components of the ECM, to improve the delivery of antitumor drugs and increase their therapeutic efficacy. The antitumor activity of paclitaxel (PTX) in HA synthase 3-overexpressing and wild-type SKOV3 ovarian cancer model and in the BxPC3 pancreas xenograft tumour model, was evaluated by monitoring tumour growth with or without PEGPH20 pre-treatment. Pharmacokinetics and tumour penetration of PTX were assessed by HPLC and mass spectrometry imaging analysis in the same tumour models. Tumour tissue architecture and HA deposition were analysed by histochemistry. RESULTS: Pre-treatment with PEGPH20 modified tumour tissue architecture and improved the antitumor activity of paclitaxel in the SKOV3/HAS3 tumour model, favouring its accumulation and more homogeneous intra-tumour distribution, as assessed by quantitative and qualitative analysis. PEGPH20 also reduced HA content influencing, though less markedly, PTX distribution and antitumor activity in the BxPC3 tumour model. CONCLUSION: Remodelling the stroma of HA-rich tumours by depletion of HA with PEGPH20 pre-treatment, is a potentially successful strategy to improve the intra-tumour distribution of anticancer drugs, increasing their therapeutic efficacy, without increasing toxicity.

A Preclinical Investigation into the Effects of Aging on Dermal Hyaluronan Properties and Reconstitution Following Recombinant Human Hyaluronidase PH20 Administration.

INTRODUCTION: There is currently no consensus in the literature concerning the impact of aging on the properties of hyaluronan (HA) in the subcutaneous (SC) space. Recombinant human hyaluronidase PH20 (rHuPH20) facilitates SC administration of injected therapeutics by depolymerizing SC HA, facilitating bulk fluid flow, dispersion and absorption. This study assessed the impact of intrinsic aging on HA in the SC space and thus the ability of rHuPH20 to enhance delivery of co-administered therapeutics. METHODS: Histologic evaluations of HA levels and degradation were performed on human skin samples from six age groups, aged from 20 to 100 years. HA levels were evaluated by HA staining and degradation by staining samples for HA following incubation with rHuPH20. HA was extracted from samples and HA size determined by gel electrophoresis. Dermal reconstitution was assessed in young (aged 1.5 months) and elderly (aged > 16 months) mice. Baseline dye dispersion was measured at 5 and 20 min post-intradermal dye injection. Following treatment with rHuPH20, dye dispersion was measured again at 2, 24, 48, 72 and 96 h. RESULTS: Distribution of HA was confined to the interstitial space between adipocytes, with similar pericellular presence and levels of HA found across all age groups. Substantial levels of high-molecular-weight HA were observed in all age groups at baseline. Incubation with a clinically relevant dose of rHuPH20 resulted in degradation of all SC HA and similar degradation profiles independent of age. No difference in dye dispersion time was observed between young and elderly mice across the range of time points assessed, with dye dispersion returning to baseline levels by 24 h after rHuPH20 treatment. CONCLUSIONS: Subcutaneous delivery of approved therapeutics facilitated by co-administration with rHuPH20 should not be impacted by intrinsic aging, with this study providing no evidence for an effect of aging on HA distribution, structure or a loss of rHuPH20 efficacy.

The growth of a xenograft breast cancer tumor model with engineered hyaluronan-accumulating stroma is dependent on hyaluronan and independent of CD44.

Hyaluronan accumulation in the tumor microenvironment is associated with poor prognosis in several solid human cancers. To understand the role of stromal hyaluronan in tumor progression, we engineered 3T3HAS3, a hyaluronan-producing fibroblast cell line, by lentiviral transduction of Balb/c 3T3 cells with the human hyaluronan synthase 3 (HAS3) gene. 3T3HAS3 cells significantly enhanced tumor growth when co-grafted with MDA-MB-468 cells in nude mice. Immunohistochemical analysis of the xenograft tumors showed that MDA-MB-468 cells were surrounded by hyaluronan-accumulating stroma, closely resembling the morphology observed in human breast cancer specimens. Tumor growth of MDA-MB-468 + 3T3HAS3 co-grafts was greatly reduced upon hyaluronan degradation by lentiviral transduction of a human hyaluronidase gene in 3T3HAS3 cells, or by systemic administration of pegvorhyaluronidase alfa (PEGPH20). In contrast, the growth of the co-graft tumors was not inhibited when CD44 expression was reduced or ablated by small hairpin RNA-mediated CD44 knockdown in MDA-MB-468 cells, CD44 CRISPR knockout in 3T3HAS3 cells, or by grafting these cells in CD44 knockout nude mice. Collectively, these data demonstrate that tumor growth of an engineered xenograft breast cancer model with hyaluronan-accumulating stroma can be dependent on hyaluronan and independent of CD44.

Dissecting the Stromal Signaling and Regulation of Myeloid Cells and Memory Effector T Cells in Pancreatic Cancer.

PURPOSE: Myeloid cells are a prominent immunosuppressive component within the stroma of pancreatic ductal adenocarcinoma (PDAC). Previously, targeting myeloid cells has had limited success. Here, we sought to target the myeloid cells through modifying a specific stromal component. EXPERIMENTAL DESIGN: A murine model of metastatic PDAC treated with an irradiated whole-cell PDAC vaccine and PDAC specimens from patients treated with the same type of vaccine were used to assess the immune-modulating effect of stromal hyaluronan (HA) degradation by PEGPH20. RESULTS: Targeting stroma by degrading HA with PEGPH20 in combination with vaccine decreases CXCL12/CXCR4/CCR7 immunosuppressive signaling axis expression in cancer-associated fibroblasts, myeloid, and CD8+ T cells, respectively. This corresponds with increased CCR7- effector memory T-cell infiltration, an increase in tumor-specific IFNγ, and improved survival. In the stroma of human PDACs treated with the same vaccine, decreased stromal CXCR4 expression significantly correlated with decreased HA and increased cytotoxic activities, suggesting CXCR4 is an important therapeutic target. CONCLUSIONS: This study represents the first to dissect signaling cascades following PDAC stroma remodeling via HA depletion, suggesting this not only overcomes a physical barrier for immune cell trafficking, but alters myeloid function leading to downstream selective increases in effector memory T-cell infiltration and antitumor activity.

Remodeling the Tumor Microenvironment Sensitizes Breast Tumors to Anti-Programmed Death-Ligand 1 Immunotherapy.

Immunotherapies targeting immune checkpoint inhibitors have changed the landscape of cancer treatment, however, many patients are resistant or refractory to immunotherapy. The sensitivity of tumor cells to immunotherapy may be influenced by hyaluronan (HA) accumulation in the tumor microenvironment (TME). Enzymatic degradation of HA by pegvorhyaluronidase alfa (PEGPH20; PVHA) remodels the TME. This leads to reduced tumor interstitial pressure and decompressed tumor blood vessels, which are both associated with increased exposure of tumor cells to chemotherapy drugs. Here, we demonstrate PVHA increased the uptake of anti-programmed death-ligand 1 (PD-L1) antibody in HA-accumulating animal models of breast cancer. The increased levels of anti-PD-L1 antibody were associated with increased accumulation of T cells and natural killer cells and decreased myeloid-derived suppressor cells. PD-L1 blockade significantly inhibited tumor growth when combined with PVHA, but not alone. Our results suggest that PVHA can sensitize HA-accumulating tumors to anti-PD-L1 immunotherapy. SIGNIFICANCE: These findings show removal of hyaluronan in the tumor microenvironment improves immune cells and checkpoint inhibitors access to tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4149/F1.large.jpg.

Dynamic Contrast-enhanced MRI Detects Responses to Stroma-directed Therapy in Mouse Models of Pancreatic Ductal Adenocarcinoma.

PURPOSE: The dense stroma underlies the drug resistance of pancreatic ductal adenocarcinoma (PDA) and has motivated the development of stroma-directed drugs. Our objective is to test the concept that dynamic contrast-enhanced (DCE) MRI using FDA-approved contrast media, an imaging method sensitive to the tumor microenvironment, can detect early responses to stroma-directed drug. EXPERIMENTAL DESIGN: Imaging studies were performed in three mouse models exhibiting high desmoplastic reactions: the autochthonous PDA in genetically engineered mice (KPC), an orthotopic model in syngeneic mice, and a xenograft model of human PDA in athymic mice. An investigational drug, PEGPH20 (pegvorhyaluronidase alfa), which degrades hyaluronan (HA) in the stroma of PDA, was injected alone or in combination with gemcitabine. RESULTS: At 24 hours after a single injection of PEGPH20, Ktrans , a DCE-MRI-derived marker that measures how fast a unit volume of contrast media is transferred from capillaries to interstitial space, increased 56% and 50% from baseline in the orthotopic and xenograft tumors, respectively, compared with a 4% and 6% decrease in vehicle groups (both P < 0.05). Similarly, after three combined treatments, Ktrans in KPC mice increased 54%, whereas it decreased 4% in controls treated with gemcitabine alone (P < 0.05). Consistently, after a single injection of PEGPH20, tumor HA content assessed by IHC was reduced substantially in all three models while drug delivery (measured by paclitaxel accumulation in tumor) was increased by 2.6-fold. CONCLUSIONS: These data demonstrated a DCE-MRI marker, Ktrans , can detect early responses to stroma-directed drug and reveal the sustained effect of combination treatment (PEGPH20+ gemcitabine).

Parallel Accumulation of Tumor Hyaluronan, Collagen, and Other Drivers of Tumor Progression.

Purpose: The tumor microenvironment (TME) evolves to support tumor progression. One marker of more aggressive malignancy is hyaluronan (HA) accumulation. Here, we characterize biological and physical changes associated with HA-accumulating (HA-high) tumors.Experimental Design: We used immunohistochemistry, in vivo imaging of tumor pH, and microdialysis to characterize the TME of HA-high tumors, including tumor vascular structure, hypoxia, tumor perfusion by doxorubicin, pH, content of collagen. and smooth muscle actin (α-SMA). A novel method was developed to measure real-time tumor-associated soluble cytokines and growth factors. We also evaluated biopsies of murine and pancreatic cancer patients to investigate HA and collagen content, important contributors to drug resistance.Results: In immunodeficient and immunocompetent mice, increasing tumor HA content is accompanied by increasing collagen content, vascular collapse, hypoxia, and increased metastatic potential, as reflected by increased α-SMA. In vivo treatment of HA-high tumors with PEGylated recombinant human hyaluronidase (PEGPH20) dramatically reversed these changes and depleted stores of VEGF-A165, suggesting that PEGPH20 may also diminish the angiogenic potential of the TME. Finally, we observed in xenografts and in pancreatic cancer patients a coordinated increase in HA and collagen tumor content.Conclusions: The accumulation of HA in tumors is associated with high tIP, vascular collapse, hypoxia, and drug resistance. These findings may partially explain why more aggressive malignancy is observed in the HA-high phenotype. We have shown that degradation of HA by PEGPH20 partially reverses this phenotype and leads to depletion of tumor-associated VEGF-A165. These results encourage further clinical investigation of PEGPH20. Clin Cancer Res; 24(19); 4798-807. ©2018 AACR.

The invadopodia scaffold protein Tks5 is required for the growth of human breast cancer cells in vitro and in vivo.

The ability of cancer cells to invade underlies metastatic progression. One mechanism by which cancer cells can become invasive is through the formation of structures called invadopodia, which are dynamic, actin-rich membrane protrusions that are sites of focal extracellular matrix degradation. While there is a growing consensus that invadopodia are instrumental in tumor metastasis, less is known about whether they are involved in tumor growth, particularly in vivo. The adaptor protein Tks5 is an obligate component of invadopodia, and is linked molecularly to both actin-remodeling proteins and pericellular proteases. Tks5 appears to localize exclusively to invadopodia in cancer cells, and in vitro studies have demonstrated its critical requirement for the invasive nature of these cells, making it an ideal surrogate to investigate the role of invadopodia in vivo. In this study, we examined how Tks5 contributes to human breast cancer progression. We used immunohistochemistry and RNA sequencing data to evaluate Tks5 expression in clinical samples, and we characterized the role of Tks5 in breast cancer progression using RNA interference and orthotopic implantation in SCID-Beige mice. We found that Tks5 is expressed to high levels in approximately 50% of primary invasive breast cancers. Furthermore, high expression was correlated with poor outcome, particularly in those patients with late relapse of stage I/II disease. Knockdown of Tks5 expression in breast cancer cells resulted in decreased growth, both in 3D in vitro cultures and in vivo. Moreover, our data also suggest that Tks5 is important for the integrity and permeability of the tumor vasculature. Together, this work establishes an important role for Tks5 in tumor growth in vivo, and suggests that invadopodia may play broad roles in tumor progression.

The induction of HIF-1 reduces astrocyte activation by amyloid beta peptide.

Reduced glucose metabolism and astrocyte activation in selective areas of the brain are pathological features of Alzheimer's disease (AD). The underlying mechanisms of low energy metabolism and a molecular basis for preventing astrocyte activation are not, however, known. Here we show that amyloid beta peptide (Abeta)-dependent astrocyte activation leads to a long-term decrease in hypoxia-inducible factor (HIF)-1alpha expression and a reduction in the rate of glycolysis. Glial activation and the glycolytic changes are reversed by the maintenance of HIF-1alpha levels with conditions that prevent the proteolysis of HIF-1alpha. Abeta increases the long-term production of reactive oxygen species (ROS) through the activation of nicotinamide adenine dinucleotide phosphate oxidase and reduces the amount of HIF-1alpha via the activation of the proteasome. ROS are not required for glial activation, but are required for the reduction in glycolysis. These data suggest a significant role for HIF-1alpha-mediated transcription in maintaining the metabolic integrity of the AD brain and identify the probable cause of the observed lower energy metabolism in afflicted areas. They may also explain the therapeutic success of metal chelators in animal models of AD.

A role for the podosome/invadopodia scaffold protein Tks5 in tumor growth in vivo.

Podosomes and invadopodia are electron-dense, actin-rich protrusions located on the ventral side of the cellular membrane. They are detected in various types of normal cells, but also in human cancer cells and in Src-transformed fibroblasts. Previously we have shown that the scaffold protein Tks5 (tyrosine kinase substrate 5) co-localizes to podosomes/invadopodia in different human cancer cells and in Src-transformed NIH-3T3 cells. Upon reduced expression of Tks5 podosome formation is decreased, which leads to diminished gelatin degradation in vitro in various human cancer cell lines. It is unclear, however, whether cancer cells need podosomes for tumor growth and metastasis in vivo. To test this idea, we evaluated the ability of Src-transformed NIH-3T3 cells, showing stable reduced expression of Tks5 and podosome formation (Tks5 KD), to form subcutaneous tumors in mice. We demonstrate that decreased expression of Tks5 correlated with reduced tumor growth at this site. In addition, we generated lung metastases from these cells following tail vein injection. The lungs of mice injected i.v. with the Tks5 KD showed smaller-sized metastases, but there was no difference in the number of lesions compared to the controls, indicating that podosomes may not be required for extravasation from the blood stream into the lung parenchyma. Independent of the microenvironment however, the reduced tumor growth correlated with decreased tumor vascularization. Our data potentially implicate a novel role of podosomes as mediators of tumor angiogenesis and support further exploration of how podosome formation and Tks5 expression contribute to tumor progression.

Brain-specific knock-out of hypoxia-inducible factor-1alpha reduces rather than increases hypoxic-ischemic damage.

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays an essential role in cellular and systemic O(2) homeostasis by regulating the expression of genes important in glycolysis, erythropoiesis, angiogenesis, and catecholamine metabolism. It is also believed to be a key component of the cellular response to hypoxia and ischemia under pathophysiological conditions, such as stroke. To clarify the function of HIF-1alpha in the brain, we exposed adult mice with late-stage brain deletion of HIF-1alpha to hypoxic injuries. Contrary to expectations, the brains from the HIF-1alpha-deficient mice were protected from hypoxia-induced cell death. These surprising findings suggest that decreasing the level of HIF-1alpha can be neuroprotective. Gene chip expression analysis revealed that, contrary to expectations, the majority of hypoxia-dependent gene-expression changes were unaltered, whereas a specific downregulation of apoptotic genes was observed in the HIF-1alpha-deficient mice. Although the role of HIF-1alpha has been extensively characterized in vitro, in cancer models, and in chronic preconditioning paradigms, this is the first study to evaluate the role of HIF-1alpha in vivo in the brain in response to acute hypoxia/ischemia. Our data suggest, that in acute hypoxia, the neuroprotection found in the HIF-1alpha-deficient mice is mechanistically consistent with a predominant role of HIF-1alpha as proapoptotic and loss of function leads to neuroprotection. Furthermore, our data suggest that functional redundancy develops after excluding HIF-1alpha, leading to the preservation of gene expression regulating the majority of other previously characterized HIF-dependent genes.

The hypoxic response of tumors is dependent on their microenvironment.

To reveal the functional significance of hypoxia and angiogenesis in astrocytoma progression, we created genetically engineered transformed astrocytes from murine primary astrocytes and deleted the hypoxia-responsive transcription factor HIF-1alpha or its target gene, the angiogenic factor VEGF. Growth of HIF-1alpha- and VEGF-deficient transformed astrocytes in the vessel-poor subcutaneous environment results in severe necrosis, reduced growth, and vessel density, whereas when the same cells are placed in the vascular-rich brain parenchyma, the growth of HIF-1alpha knockout, but not VEGF knockout tumors, is reversed: tumors deficient in HIF-1alpha grow faster, and penetrate the brain more rapidly and extensively. These results demonstrate that HIF-1alpha has differential roles in tumor progression, which are greatly dependent on the extant microenvironment of the tumor.