Mild Hyperthermia Enhanced Liposomal Doxorubicin Delivery and CD8+ T cell Infiltration in Triple Negative Breast Cancer.

Mild hyperthermia (MHTh) is often used in combination with chemotherapy and radiotherapy for cancer treatment. In the current study, the effect of MHTh on the enhanced uptake of the FDA-approved chemotherapy drug, liposomal doxorubicin (dox) in syngeneic 4T1 tumors was investigated. Doxorubicin has inherent fluorescence properties having an emission signal at 590 nm upon excitation with a 480 nm laser. A group of mice administered with doxorubicin (dox) were exposed to MHTh (42 °C) for 30 minutes whereas control group given dox did not receive MHTh. Ex vivo optical imaging of harvested tumors confirmed higher uptake of dox in treated versus the control untreated tumors. Confocal microscopy of tumor sections indicates higher fluorescent intensity due to increased accumulation of dox in MHTh-treated compared to untreated tumors. We examined the effect of MHTh to enhance CD8 tumor infiltration, production of interferon-γ (IFN-γ) and expression of programmed death ligand-1 (PD-L1). mRNA in situ hybridization was performed to test for transcripts of CD8, IFN-γ and PD-L1. Results showed that higher expression of CD8 mRNA was observed in MHTh-administered tumors versus untreated cohorts. The signal for IFN-γ and PD-L1 in both groups were not significantly different. Taken together, our findings imply that MHTh can improve tumor uptake of dox. Importantly, our data suggests that MHTh can boost CD8+ T cell infiltration.

Detection of IL12/23p40 via PET Visualizes Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), which includes both Crohn disease and ulcerative colitis, is a relapsing inflammatory disease of the gastrointestinal tract. Long-term chronic inflammatory conditions elevate the patient's risk of colorectal cancer (CRC). Currently, diagnosis requires endoscopy with biopsy. This procedure is invasive and requires a bowel-preparatory regimen, adding to patient burden. Interleukin 12 (IL12) and interleukin 23 (IL23) play key roles in inflammation, especially in the pathogenesis of IBD, and are established therapeutic targets. We propose that imaging of IL12/23 and its p40 subunit in IBD via immuno-PET potentially provides a new noninvasive diagnostic approach. Methods: Our aim was to investigate the potential of immuno-PET to image inflammation in a chemically induced mouse model of colitis using dextran sodium sulfate by targeting IL12/23p40 with a 89Zr-radiolabeled anti-IL12/23p40 antibody. Results: High uptake of the IL12/23p40 immuno-PET agent was exhibited by dextran sodium sulfate-administered mice, and this uptake correlated with increased IL12/23p40 present in the sera. Competitive binding studies confirmed the specificity of the radiotracer for IL12/23p40 in the gastrointestinal tract. Conclusion: These promising results demonstrate the utility of this radiotracer as an imaging biomarker of IBD. Moreover, IL12/23p40 immuno-PET can potentially guide treatment decisions for IBD management.

Selective ablation of TRA-1-60+ pluripotent stem cells suppresses tumor growth of prostate cancer.

Purpose: TRA-1-60 (TRA) is an established transcription factor of embryonic signaling and a well-known marker of pluripotency. It has been implicated in tumorigenesis and metastases, is not expressed in differentiated cells, which makes it an appealing biomarker for immunopositron emission tomography (immunoPET) imaging and radiopharmaceutical therapy (RPT). Herein, we explored the clinical implications of TRA in prostate cancer (PCa), examined the potential of TRA-targeted PET to specifically image TRA+ cancer stem cells (CSCs) and assessed response to the selective ablation of PCa CSCs using TRA-targeted RPT. Experimental Design: First, we assessed the relationship between TRA (PODXL) copy number alterations (CNA) and survival using publicly available patient databases. The anti-TRA antibody, Bstrongomab, was radiolabeled with Zr-89 or Lu-177 for immunoPET imaging and RPT in PCa xenografts. Radiosensitive tissues were collected to assess radiotoxicity while excised tumors were examined for pathologic treatment response. Results: Patients with tumors having high PODXL CNA exhibited poorer progression-free survival than those with low PODXL, suggesting that it plays an important role in tumor aggressiveness. TRA-targeted immunoPET imaging specifically imaged CSCs in DU-145 xenografts. Tumors treated with TRA RPT exhibited delayed growth and decreased proliferative activity, marked by Ki-67 immunohistochemistry. Aside from minor weight loss in select animals, no significant signs of radiotoxicity were observed in the kidneys or livers. Conclusions: We successfully demonstrated the clinical significance of TRA expression in human PCa, engineered and tested radiotheranostic agents to image and treat TRA+ prostate CSCs. Ablation of TRA+ CSCs blunted PCa growth. Future studies combining CSC ablation with standard treatment will be explored to achieve durable responses.

Evaluation and selection of a lead diabody for interferon-γ PET imaging.

INTRODUCTION: Interferon-γ (IFN-γ) is an appealing target to evaluate immune response in cancer immunotherapy as it is a hallmark of an active immune system. Imaging and detection via immunopositron emission tomography (immunoPET) of this soluble cytokine has been made feasible using a 89Zr-labeled (t 1/2 ~ 3.27 d) monoclonal antibody (mAb). Because of its size, using a full-length mAb as an imaging vector is not ideal for repeat serial imaging because of its prolonged blood pool residency and tumor accumulation resulting in lengthier wait times between administration and imaging. This consequently impacts the potential to image a dynamic immune response in real time. This work compares 89Zr-labeled diabodies (Db) designed with variable linker lengths between the VH and VL regions with the goal of selecting a lead Db for future studies. METHODS AND RESULTS: Four Db fragments with various linker lengths (HL-n, n = 7-13 amino acids) were each conjugated to desferrioxamine (DFO). The number of attached chelates was analyzed via mass spectrometry with all immunoconjugates exhibiting one unit of DFO attached. Db-DFO conjugates were subsequently radiolabeled with zirconium-89. All constructs radiolabeled with high yields. Each radioimmunoconjugate was tested for reactivity to IFN-γ. All tracers except for [89Zr]Zr-DFO-NCS-anti-IFN-γ HL-9 exhibited comparable immunoreactivities (>90 %) to the radiolabeled parent mAb (95.8 %). At 24 h post-labeling, the IRF values were retained except for the HL-13 construct. Imaging scans and tissue distribution studies acquired in mice bearing CT26 syngeneic colorectal tumors between 1 and 24 h post-tracer administration demonstrated variable clearance kinetics and tumor localization of each radiotracer. HL-7 had higher binding in non-tumor tissues compared to HL-11 and HL-13 at 3 h p.i. Competitive binding studies versus unmodified parent mAb (AN-18) demonstrated blocking of radiolabeled HL-11 and HL-13. [89Zr]Zr-DFO-NCS-anti-IFN-γ HL-7 was inadequately blocked. CONCLUSION: Despite nuanced differences in linker lengths, our data demonstrates that [89Zr]Zr-DFO-NCS-anti-IFN-γ HL-11 exhibited the best radiotracer properties for the assessment of IFN-γ production in vivo. Work is currently underway to test the potential of using shorter-lived isotopes, like copper-64 (t1/2 ~ 12.7 h) to match pharmacokinetics and half-lives.

p62/SQSTM1-induced caspase-8 aggresomes are essential for ionizing radiation-mediated apoptosis.

The autophagy-lysosome pathway and apoptosis constitute vital determinants of cell fate and engage in a complex interplay in both physiological and pathological conditions. Central to this interplay is the archetypal autophagic cargo adaptor p62/SQSTM1/Sequestosome-1 which mediates both cell survival and endoplasmic reticulum stress-induced apoptosis via aggregation of ubiquitinated caspase-8. Here, we investigated the role of p62-mediated apoptosis in head and neck squamous cell carcinoma (HNSCC), which can be divided into two groups based on human papillomavirus (HPV) infection status. We show that increased autophagic flux and defective apoptosis are associated with radioresistance in HPV(-) HNSCC, whereas HPV(+) HNSCC fail to induce autophagic flux and readily undergo apoptotic cell death upon radiation treatments. The degree of radioresistance and tumor progression of HPV(-) HNSCC respectively correlated with autophagic activity and cytosolic levels of p62. Pharmacological activation of the p62-ZZ domain using small molecule ligands sensitized radioresistant HPV(-) HNSCC cells to ionizing radiation by facilitating p62 self-polymerization and sequestration of cargoes leading to apoptosis. The self-polymerizing activity of p62 was identified as the essential mechanism by which ubiquitinated caspase-8 is sequestered into aggresome-like structures, without which irradiation fails to induce apoptosis in HNSCC. Our results suggest that harnessing p62-dependent sequestration of ubiquitinated caspase-8 provides a novel therapeutic avenue in patients with radioresistant tumors.

Discoidin Domain Receptors, DDR1b and DDR2, Promote Tumour Growth within Collagen but DDR1b Suppresses Experimental Lung Metastasis in HT1080 Xenografts.

The Discoidin Domain Receptors (DDRs) constitute a unique set of receptor tyrosine kinases that signal in response to collagen. Using an inducible expression system in human HT1080 fibrosarcoma cells, we investigated the role of DDR1b and DDR2 on primary tumour growth and experimental lung metastases. Neither DDR1b nor DDR2 expression altered tumour growth at the primary site. However, implantation of DDR1b- or DDR2-expressing HT1080 cells with collagen I significantly accelerated tumour growth rate, an effect that could not be observed with collagen I in the absence of DDR induction. Interestingly, DDR1b, but not DDR2, completely hindered the ability of HT1080 cells to form lung colonies after intravenous inoculation, suggesting a differential role for DDR1b in primary tumour growth and lung colonization. Analyses of tumour extracts revealed specific alterations in Hippo pathway core components, as a function of DDR and collagen expression, that were associated with stimulation of tumour growth by DDRs and collagen I. Collectively, these findings identified divergent effects of DDRs on primary tumour growth and experimental lung metastasis in the HT1080 xenograft model and highlight the critical role of fibrillar collagen and DDRs in supporting the growth of tumours thriving within a collagen-rich stroma.

The mouse prostate: a basic anatomical and histological guideline.

Despite substantial similarities in embryological, cellular and molecular biology features, human and mouse prostates differ in organ morphology and tissue architecture. Thus, a clear understanding of the anatomy and histology of the mouse prostate is essential for the identification of urogenital phenotypes in genetically engineered mice, as well as for the study of the etiology, development, and treatment of human prostatic diseases for which mouse models are used. The purpose of this manuscript is to provide a brief guide for the dissection of the mouse prostate and the identification of its different lobes and histology, to both basic researchers and medical pathologists who are unfamiliar with mouse tissues.

Bone-induced c-kit expression in prostate cancer: a driver of intraosseous tumor growth.

Loss of BRCA2 function stimulates prostate cancer (PCa) cell invasion and is associated with more aggressive and metastatic tumors in PCa patients. Concurrently, the receptor tyrosine kinase c-kit is highly expressed in skeletal metastases of PCa patients and induced in PCa cells placed into the bone microenvironment in experimental models. However, the precise requirement of c-kit for intraosseous growth of PCa and its relation to BRCA2 expression remain unexplored. Here, we show that c-kit expression promotes migration and invasion of PCa cells. Alongside, we found that c-kit expression in PCa cells parallels BRCA2 downregulation. Gene rescue experiments with human BRCA2 transgene in c-kit-transfected PCa cells resulted in reduction of c-kit protein expression and migration and invasion, suggesting a functional significance of BRCA2 downregulation by c-kit. The inverse association between c-kit and BRCA2 gene expressions in PCa cells was confirmed using laser capture microdissection in experimental intraosseous tumors and bone metastases of PCa patients. Inhibition of bone-induced c-kit expression in PCa cells transduced with lentiviral short hairpin RNA reduced intraosseous tumor incidence and growth. Overall, our results provide evidence of a novel pathway that links bone-induced c-kit expression in PCa cells to BRCA2 downregulation and supports bone metastasis.

Maspin expression in prostate tumor elicits host anti-tumor immunity.

The goal of the current study is to examine the biological effects of epithelial-specific tumor suppressor maspin on tumor host immune response. Accumulated evidence demonstrates an anti-tumor effect of maspin on tumor growth, invasion and metastasis. The molecular mechanism underlying these biological functions of maspin is thought to be through histone deacetylase inhibition, key to the maintenance of differentiated epithelial phenotype. Since tumor-driven stromal reactivities co-evolve in tumor progression and metastasis, it is not surprising that maspin expression in tumor cells inhibits extracellular matrix degradation, increases fibrosis and blocks hypoxia-induced angiogenesis. Using the athymic nude mouse model capable of supporting the growth and progression of xenogeneic human prostate cancer cells, we further demonstrate that maspin expression in tumor cells elicits neutrophil- and B cells-dependent host tumor immunogenicity. Specifically, mice bearing maspin-expressing tumors exhibited increased systemic and intratumoral neutrophil maturation, activation and antibody-dependent cytotoxicity, and decreased peritumoral lymphangiogenesis. These results reveal a novel biological function of maspin in directing host immunity towards tumor elimination that helps explain the significant reduction of xenograft tumor incidence in vivo and the clinical correlation of maspin with better prognosis of several types of cancer. Taken together, our data raised the possibility for novel maspin-based cancer immunotherapies.

PTEN loss mediated Akt activation promotes prostate tumor growth and metastasis via CXCL12/CXCR4 signaling.

INTRODUCTION: The chemokine CXCL12, also known as SDF-1, and its receptor, CXCR4, are overexpressed in prostate cancers and in animal models of prostate-specific PTEN deletion, but their regulation is poorly understood. Loss of the tumor suppressor PTEN (phosphatase and tensin homolog) is frequently observed in cancer, resulting in the deregulation of cell survival, growth, and proliferation. We hypothesize that loss of PTEN and subsequent activation of Akt, frequent occurrences in prostate cancer, regulate the CXCL12/CXCR4 signaling axis in tumor growth and bone metastasis. METHODS: Murine prostate epithelial cells from PTEN+/+, PTEN+/-, and PTEN-/- (prostate specific knockdown) mice as well as human prostate cancer cell lines C4-2B, PC3, and DU145 were used in gene expression and invasion studies with Akt inhibition. Additionally, HA-tagged Akt1 was overexpressed in DU145, and tumor growth in subcutaneous and intra-tibia bone metastasis models were analyzed. RESULTS: Loss of PTEN resulted in increased expression of CXCR4 and CXCL12 and Akt inhibition reversed expression and cellular invasion. These results suggest that loss of PTEN may play a key role in the regulation of this chemokine activity in prostate cancer. Overexpression of Akt1 in DU145 resulted in increased CXCR4 expression, as well as increased proliferation and cell cycle progression. Subcutaneous injection of these cells also resulted in increased tumor growth as compared to neo controls. Akt1 overexpression reversed the osteosclerotic phenotype associated with DU145 cells to an osteolytic phenotype and enhanced intra-osseous tumor growth. CONCLUSIONS: These results suggest the basis for activation of CXCL12 signaling through CXCR4 in prostate cancer driven by the loss of PTEN and subsequent activation of Akt. Akt1-associated CXCL12/CXCR4 signaling promotes tumor growth, suggesting that Akt inhibitors may potentially be employed as anticancer agents to target expansion of PC bone metastases.